Dirigent gene EG261 and its orthologs and paralogs and their uses for pathogen resistance in plants

ABSTRACT

The present invention provides the identification and use of EG261, homologs of EG261, orthologs of EG261, paralogs of EG261, and fragments and variations thereof for altering, e.g. increasing, pathogen tolerance and/or resistance in plants.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a continuation-in-part of international PCT application No.PCT/US2013/042382 filed on May 23, 2013 and U.S. non-provisionalapplication Ser. No. 13/901,071 filed on May 23, 2013, both of whichclaim priority to U.S. provisional application No. 61/789,463 filed onMar. 15, 2013 and U.S. provisional application No. 61/652,029 filed onMay 25, 2012 each of which is hereby incorporated by reference in itsentirely for all purposes.

DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY

The contents of the text file submitted electronically herewith areincorporated herein by reference in their entirety: A computer readableformat copy of the Sequence Listing (filename:EVOL_004_04US_SeqList_ST25.txt, date recorded: Sep. 3, 2014, file size89 kilobytes).

TECHNICAL FIELD

The invention relates to the identification and use of nucleic acidsequences for pathogen resistance in plants.

BACKGROUND

“Dirigent” refers to genes or proteins which are members of a gene orprotein family, respectively, members of which have been identified inmany plants. Dirigents have been implicated in resistance to varioustypes of pathogens in a range of different, and sometimes distantlyrelated, plants.

Dirigent proteins confer a broad response to many pathogens in a numberof plants species, including for conifers (Ralph et al., Plant MolecularBiology (2006) 60:21-40); cotton (L. Zhu, X. Zhang, L. Tu, F. Zeng, Y.Nie and X. Guo Journal of Plant Pathology, 2007. 89 (1), 41-45); barley(Ralph et al., Plant Molecular Biology, 2006, 60:21-40_DOI10.1007/s11103-005-2226-y), barley, (Kristensen et al. Plant Physiology,June 1999, Vol. 120, pp. 501-512): orange trees (“Gene expression inCitrus sinensis (L.) Osbeck following infection with the bacterialpathogen Candidatus Liberibacter asiatiens causing Huanglongbing inFlorida” Albrecht et al., Plant Science, Volume 175, Issue 3, September2008, Pages 291-304 wheat (poster presentation: “Cloning andTranscriptional Profiling of a Dirigent-like Gene from Wheat Respondingto Hessian Fy Infestation”, C. Williams, Poster PO910, Plant and AnimalGenome 20): and pea (“Transgenic canola lines expressing pea defensegene DRR206 have resistance to aggressive blackleg isolates and toRhizoctonia solanil,”, Wang and Fristensky, Molecular Breeding Volume 8,Number 3 (2001), 263-271, DOI: 10.1023/A:1013706400168). Thus, dirigentshave been implicated in pathogen response in many plants, and thisresponse has been shown for a number of different pathogens, includingbut not limited to fungi, bacteria, insects and nematodes.

The identification and use of dirigents is important to plant husbandryand crop production, particularly for commercial crop production inagronomy and horticulture.

SUMMARY OF THE INVENTION

The present invention provides the identification and use of EG261,homologs of EG261, orthologs of EG261, paralogs of EG261, and fragmentsand variations thereof for altering, e.g. conferring or increasing,pathogen tolerance and/or resistance in plants. Importantly, suchpathogen altered, e.g., increased, tolerance and/or resistance can beobtained using conventional plant breeding methods, whereby such methodsoptionally also include using any of various biotechnological methodsfor verifying that the desired EG261, homologs of EG261, orthologs ofEG261, paralogs of EG261, and fragments and variations thereof arepresent in the resulting crosses and offspring.

In embodiments, the present invention provides isolated nucleic acidsequences coding for EG261, homologs of EG261, orthologs of EG261,paralogs of EG261, and fragments and variations thereof. The presentinvention also provides chimeric genes, constructs, recombinant DNA,vectors, plant cells, plant tissues, plant parts, plant tissue culturesand/or whole plants comprising such nucleic acid sequences.

In one embodiment, the present invention provides polynucleotides foraltering and/or increasing pathogen tolerance and/or resistancecomprising a nucleic acid sequence that shares at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95% at least96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%,at least 99.7%, at least 99.8%, or at least 99.9% identity to nucleicacids coding for EG261, homologs of EG261, orthologs of EG261, paralogsof EG261, and fragments and variations thereof.

The present invention further provides isolated amino acid sequences(e.g., a peptide, polypeptide and the like) comprising an amino acidsequence encoded by the nucleic, acid sequences for EG261, homologs ofEG261, orthologs of EG261, paralogs of EG261, and fragments andvariations thereof.

In some embodiments, the present invention provides isolated amino acidsequences which form a protein that shares an amino acid sequence havingat least 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, atleast 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9%identity to an amino acid sequence encoded by the nucleic acid sequencesfor EG261, homologs of EG261, orthologs of EG261 paralogs of EG261, andfragments and variations thereof.

In one embodiment, the present invention provides isolated amino acidsequences which form a protein that shares an amino acid having at least85%, at least 86%, at least 87%, at least 88%, at least 89%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, at least 99%, at least99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%,at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9%identity to an amino acid sequence encoded by the nucleic acid sequencesfor EG261, homologs of EG261 orthologs of EG261, paralogs of EG261, andfragments and variations thereof.

The present invention also provides a chimeric gene comprising theisolated nucleic acid sequence of any one of the polynucleotidesdescribed above operably linked to suitable regulatory sequences.

The present invention also provides a recombinant construct comprisingthe chimeric genes as described above.

The present invention further comprises interfering RNA (RNAi) based onthe expression of the nucleic acid sequences of the present invention,wherein such RNAi includes but is not limited to microRNA (miRNA) andsmall interfering RNA (siRNA) which can be used in gene silencingconstructs.

The present invention also provides transfigured host cells comprisingthe chimeric genes as described above. In one embodiment, said hostcells are selected from the group consisting of bacteria, yeasts,filamentous fungi, algae, animals, and plants.

The present invention in another aspect provides plants comprising inits genome one or more genes as described herein, one or more genes withimitations as described herein, or the chimeric genes as describedherein. In some embodiments, the plant is derived from a soybean varietysensitive to soybean cyst nematode (SCN), and wherein the gene comprisesnucleic acid sequence encoding an EG261 ortholog derived from G.pescadrensis.

The present invention in another aspect provides plant seed obtainedfrom the plants described herein, wherein the plants producing suchseeds comprise in their genomes one or more genes as described herein,one or more genes with imitations as described herein, or the chimericgenes as described herein.

In some embodiments, the methods comprise introducing mutations in oneor more nucleic acid sequences for EG261, homologs of EG261, orthologsof EG261, paralogs of EG261 and fragments and variations thereof.

In one aspect, the present invention provides methods of breeding plantsto alter, e.g. confer or increase, pathogen tolerance and/or resistance.In one embodiment, such methods comprise making a cross between a firstplant comprising one or more nucleic acid sequences for EG261, homologsof EG261, orthologs of EG261, paralogs of EG261, and fragments andvariations with a second plant of the same or different species toproduce an F1 plant; backcrossing the F1 plant to the second plant; andrepeating the backcrossing step to generate a near isogenic line,wherein the one or more nucleic, acid sequences for EG261, homologs ofEG261, orthologs of EG261, paralogs of EG261, and fragments andvariations thereof are integrated into the genome of the second plant;wherein the near isogenic line derived from the second plant hasaltered, e.g. increased, pathogen tolerance and/or resistance.Optionally, such methods can be facilitated by using variousbiotechnological methods to verify that the nucleic acid sequences forEG26:1, homologs of EG261, orthologs of EG261, paralogs of EG261, andfragments and variations thereof are included in the second plant. Insome embodiments, the first plant comprises a nucleic acid sequenceencoding an EG261 ortholog derived from G. pescadrensis. In someembodiments, the second plant is a soybean variety sensitive to soybeancyst nematode (SCN).

The present invention provides isolated polynucleotides comprising anucleic acid sequence selected from the group consisting of (a) anucleic acid sequence having at least 95% identical nucleotides to anucleic acid sequence coding for EG261; (b) a nucleic acid sequencehaving at least 95% identical nucleotides to a nucleic acid sequencecoding for a homolog of EG261; (c) a nucleic acid sequence having atleast 95% identical nucleotides to a nucleic acid sequence coding for anortholog of EG261; (d) a nucleic acid sequence having at least 95%identical nucleotides to a nucleic acid sequence coding for a paralog ofEG261; (e) complements of a nucleic acid sequence of (a), (b), (c) or(d); (f) reverse complements of a nucleic acid sequence of (a), (c) or(d); (g) reverse sequences of a nucleic acid sequence of (a), (b), or(d); (h) mRNA sequences of a nucleic acid sequence of (a), (b), (c) or(d); and, (i) fragments and variations of a nucleic acid sequence of(a), (b), (c). (d), (e), (f), (g), and (h).

The present invention provides vectors comprising one or more of thepolynucleotides described herein.

The present invention also provides genetic constructs comprising one ormore of the isolated polynucleotides described herein.

The present invention also provides genetic constructs comprising, inthe 5′-3′ direction: (a) a promoter sequence, (b) one or more of theisolated polynucleotides described herein; and (c) a gene terminationsequence.

In some embodiments of the present invention the genetic constructscomprise an open reading frame encoding a polypeptide capable ofaltering pathogen tolerance and/or resistance.

In some embodiments of the present invention the pathogen toleranceand/or resistance is conferred or enhanced.

In some embodiments of the present invention the pathogen is soybeancyst nematode.

The present invention further provides transgenic cells comprising thegenetic constructs described herein.

The present invention also provides organisms comprising the transgeniccells described herein.

In some embodiments of the present invention the organism is a plant. Insome specific embodiments of the present invention the plant is asoybean plant.

The present invention provides plants and progeny plants thereof havingaltered pathogen tolerance and/or resistance as a result of inheritingthe polynucleotides described herein.

The present invention provides methods of producing hybrid seed whichinclude crossing the plants and progeny plants described herein with adifferent plant of the same species, and harvesting the resultant seed.

The present invention also provides methods for modifying geneexpression in a target organism comprising stably incorporating into thegenome of the organism a genetic construct described herein. In somesuch embodiments of the present invention the organism is a plant. Insome specific embodiments the plant is a soybean plant, a garden bean,or a cowpea plant.

The present invention provides methods for producing a plant havingaltered pathogen tolerance and/or resistance comprising: (a)transforming a plant cell with a genetic construct to provide atransgenic cell, wherein the genetic construct comprises: (i) a promotersequence; (ii) an isolated polynucleotide sequence of the presentinvention as described herein; and (iii) a gene termination sequence;and (b) cultivating the transgenic cell under conditions conducive toregeneration and mature plant growth of a plant having altered pathogentolerance and/or resistance. In some such embodiments the plant cell isa soybean plant cell, a garden bean plant cell, or a cowpea plant cell.

The present invention also provides methods for modifying a phenotype ofa target organism comprising stably incorporating into the genome of thetarget organism a genetic construct comprising: (a) a promoter sequence;(b) an isolated polynucleotide sequence of the present invention asdescribed herein; and (c) a gene termination sequence. In some suchembodiments the target organism is a plant. In some specific embodimentsthe plant is a soybean plant, a garden bean plant, or a cowpea plant.

The present invention provides processes of determining the presence orabsence of a polynucleotide coding for EG261, homologs of EG261,orthologs of EG261 paralogs of EG261, and fragments and variationsthereof in a plant, wherein the process comprises at least one of (a)isolating nucleic acid molecules from said plant and amplifyingsequences homologous to the polynucleotide; (b) isolating nucleic acidmolecules from said plant and performing a Southern hybridization todetect the polynucleotide; (c) isolating proteins from said plant andperforming a Western Blot using antibodies to a protein encoded by thepolynucleotide; and/or (d) demonstrating the presence of in RNAsequences derived from a polynucleotide mRNA transcript and unique tothe polynucleotide.

The present invention provides methods of breeding plants to producealtered pathogen tolerance and/or resistance comprising: i) making across between a first plant with an isolated polynucleotide sequence ofclaim 1 with a second plant to produce a F1 plant; ii) backcrossing theF1 plant to the second plant; and iii) repeating the backcrossing stepto generate a near isogenic or isogenic line, wherein the isolatedpolynucleotide sequence of claim 1 is integrated into the genome of thesecond plant and the near isogenic or isogenic line derived from thesecond plant with the isolated polynucleotide sequence has conferred orenhanced pathogen tolerance and/or resistance compared to that of thesecond plant without the isolated polynucleotide sequence. In some suchembodiments of the present invention the plant is soybean. In some suchembodiments of the present invention the pathogen is soybean cystnematode. In some embodiments, the first plant comprises a nucleic acidsequence encoding an EG261 ortholog derived from G. pescadrensis. Insome embodiments, the second plant is a soybean variety sensitive tosoybean cyst nematode (SCN).

The present invention provides methods of producing a plant withconferred or enhanced pathogen tolerance and/or resistance, the processcomprising: (a) crossing a first plant containing a polynucleotidecoding for EG261, homologs of EG261, orthologs of EG261, paralogs ofEG261, and fragments and variations thereof to a second plant, andharvesting the resultant seed; (b) determining the presence of thepolynucleotide in the resultant seed or in cells or tissues of a plantgrown from the resultant seed; wherein the determining comprises atleast one of: (i) isolating nucleic acid molecules from the resultantseed or in cells or tissues of a plant grown from the resultant seed andamplifying sequences homologous to the polynucleotide; (ii) isolatingnucleic acid molecules from the resultant seed or in cells or tissues ofa plant grown from the resultant seed and performing a Southernhybridization to detect the polynucleotide; (iii) isolating proteinsfrom the resultant seed or in cells or tissues of a plant grown from theresultant seed and performing a Western Blot using antibodies to aprotein encoded by the polynucleotide; and/or (iv) demonstrating thepresence in the resultant seed or in cells or tissues of a plant grownfrom the resultant seed of mRNA sequences derived from a polynucleotidemRNA transcript and unique to the polynucleotide. In some suchembodiments the method further comprises confirming that the resultantseed or the cells or tissues of the plant grown from the resultant seedcontain the polynucleotide. In some such embodiments the methods furthercomprise using the resultant plant containing the polynucleotide in aplant breeding scheme. In some embodiments the methods further comprisecrossing the resultant plant containing the polynucleotide with anotherplant of the same species. In some embodiments, the first plantcomprises a nucleic acid sequence encoding an EG261 ortholog derivedfrom G. pescadrensis. In some embodiments, the second plant is a soybeanvariety sensitive to soybean cyst nematode (SCN).

The present invention also provides methods of introducing the EG261orthologs conferring nematode resistance isolated according to thepresent invention into a nematode-susceptible plant of the same ordifferent species in order to increase nematode resistance of the plantand the plants produced by such methods.

The present invention also provides methods of introducing the EG261orthologs of the present invention into nematode-resistant plants of thesame or different species in order to further increase nematoderesistance of the plant and the plants produced by such methods. Forexample, according to the present invention, a gene encoding any one ofthe proteins of SEQ ID NOs: 9-1.5, 25, 26, 27, 28, 29, 30, 31, 32, 33,37, 38, 39, 43, 44, 45, 51, 52, 53, 54, 55, 57, 59, 61, 63, 64, or 65can be introduced into nematode resistant plants containing any nematoderesistant traits such as rhg1, rhg4, or any other known QTLs such asthose listed in Concibido et al., 2004 (A decade of QTL mapping for cystnematode resistance in soybean, Crop Sci. 14: 1121-1131); Shannon et al.(2004) Breeding for resistance and tolerance. In: Biology and managementof soybean cyst nematode, 2^(nd) ed, P. Schmitt, 3. A. Wrather, and R.D. Riggs, (editors). Pg 155-180: and Klink et al., (2013) EngineeredSoybean Cyst Nematode Resistance, In: Soybean-Pest Resistance, Hany A.El-Shemy (editor), pg-139-172.

The present invention also provides incorporating nematode resistanceinto a plant via non-transgenic (i.e., traditional) methods such asplant breeding and the plants produced by such methods. For example,according to the present invention, a cross can be made between a firstplant with a naturally-occurring or transgenic EG261 gene copyconferring nematode resistance, and a second plant to produce a F1plant. This F1 plant can then be subjected to multiple backcrossings togenerate a near isogenic or isogenic line, wherein the nematoderesistant copy of EG261 is integrated into the genome of the secondplant.

The EG261, homologs of EG261, orthologs of EG261, paralogs of EG261,and/or fragments and variations thereof of the present invention providebroad pathogen resistance in many, distantly related plant species. Forexample, orthologs of EG261 may be effective in a number of commerciallyimportant crop species, including, e.g., soybeans, garden beans,cowpeas, and cotton.

In some embodiments, the present invention teaches a transgenic soybeanplant, plant part, plant cell, or plant tissue culture comprising aconstruct comprising a nucleic acid sequence encoding an EG261polypeptide having at least 97% sequence identity to SEQ ID NO: 12;wherein said transgenic soybean plant, or a transgenic soybean plantproduced from said transgenic plant part, plant cell, or plant tissueculture expresses said EG261 polypeptide, and has enhanced toleranceand/or resistance to soybean cyst nematodes (SCN) compared tountransformed control soybean plants, and wherein the construct furthercomprises a EG261 native promoter operably linked to said nucleic acidencoding the EG261 polypeptide.

In some embodiments, the EG261 native promoter is selected from thegroup consisting of SEQ ID NOs: 69 and 70.

Ii some embodiments, the construct comprising the EG261 native promoteroperably linked to the nucleic acid encoding the EG261 polypeptidefurther comprises a gene termination sequence.

In some embodiments, the construct comprising the EG261 native promoterof SEQ ID NO: 69 or 70 operably linked to the nucleic acid encoding theEG261 polypeptide further comprises a gene termination sequence.

In some embodiments the gene termination sequence is a nopaline synthase(NOS) terminator.

In some embodiments, the present invention teaches a method forproducing a transgenic soybean plant having enhanced tolerance and/orresistance to soybean cyst nematode, said method comprising: (i)transforming a soybean plant cell with a construct comprising a nucleicacid sequence encoding an EG261 polypeptide having at least 97% identityto SEQ ID NO: 12; and (ii) cultivating the transgenic soybean cell underconditions conducive to regeneration and mature plant growth; whereinthe transgenic soybean plant regenerated from said transgenic plant cellexpresses said EG261 polypeptide, and has enhanced tolerance and/orresistance to soybean cyst nematodes compared to untransformed controlsoybean plants, and wherein the construct further comprises a EG261native promoter operably linked to said nucleic acid encoding, the EG261polypeptide.

In some embodiments, the EG261 native promoter is selected from thegroup consisting of SEQ ID NOs: 69 and 70.

In some embodiments, the present invention teaches a method of producinghybrid soybean seed, said method comprising crossing the transgenicsoybean plant expressing the EG261 polypeptide with the EG261 nativepromoter as described above with another soybean plant, and harvestingthe resultant seed.

In some embodiments, the present invention also teaches the progenyplants produced by the crosses between the transgenic soybean plantexpressing the EG261 polypeptide with the EG261 native promoter asdescribed above with another soybean plant as described above, whereinthe progeny soybean plants have enhanced tolerance and/or resistance tosoybean cyst nematodes compared to untransformed control soybean plantsas a result of inheriting the nucleic acid encoding the EG261 gene.

In some embodiments, the present invention teaches a method of breedingsoybean plants to produce plants with enhanced tolerance and/orresistance to soybean cyst nematodes, said method comprising: (i) makinga cross between a first transgenic soybean plant with a nucleic acidencoding a EG261 polypeptide operably linked to a EG261 native promoteras described above with a second plant to produce an F1 plant; (ii)backcrossing the F1 plant to the second plant; and (iii) repeating thebackcrossing step one or more times to generate a near isogenic orisogenic line, wherein the construct comprising the EG261 gene andpromoter is integrated into the genome of the second plant and the nearisogenic or isogenic line derived from the second plant with the nucleicacid sequence encoding the EG261 polypeptide has enhanced pathogentolerance and/or resistance to soybean cyst nematodes compared to thatof the second plant without said nucleic acid sequence.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts results showing that silencing of EG261 reduces the SCNresistance of “Fayette”, a SCN-resistant variety of soybean.

FIG. 2 depicts sequence alignments of dirigent proteins encoded by thedirigent genes of SEQ ID NOs 9 to 15.

FIG. 3 depicts sequence alignments of dirigent proteins encoded by thedirigent genes of Solanaceae family plant species.

FIG. 4 depicts sequence alignments of dirigent proteins encoded by thedirigent genes of monocot plant species.

FIG. 5 depicts sequence alignments of the core promoter sequences forthe G. pescadrensis and G. max EG261 dirigent genes. TATA boxpredictions are underlined.

DETAILED DESCRIPTION Definitions

The term “a” or “an” refers to one or more of that entity; for example,“a gene” refers to one or more genes or at least one gene. As such, theterms “a” (or “an”), “one or more” and “at least one” are usedinterchangeably herein. In addition, reference to “an element” by theindefinite article “a” or “an” does not exclude the possibility thatmore than one of the elements is present, unless the context clearlyrequires that there is one and only one of the elements.

As used herein, the verb “comprise” as is used in this description andin the claims and its conjugations are used in its non-limiting sense tomean that items following the word are included, but items notspecifically mentioned are not excluded.

As used herein, the term “plant” refers to any living organism belongingto the kingdom Plantae (i.e., any genusspecies in the Plant Kingdom),including but not limited to, Glycine spp. (e.g. soybean). Solanaceaespecies (e.g. Solanum lycopersicum, Solanum chmielewskii, Solanumhabrachaites, Solanum corneliomuller, Capsicum annuum, Solanummelongena, Solanum tubersum), Phaseolus vulgaris, Vigna unguiculata,Coffee arabica, Zen mays, Sorghum spp., Oryza sativa, Triticum spp.Hordeum spp., Gossypium hirsutum, and Heliotopium curassavicum.

As used herein, the term “plant part” refers to any part of a plantincluding but not limited to the shoot, toot; stem, seeds, fruits,stipules, leaves, petals, flowers, ovules, bracts, branches, petioles,internodes, bark, pubescence, tillers, rhizomes, fronds, blades, pollen,stamen, rootstock, scion and the like. The two main parts of plants gownin some sort of media, such as soil, are often referred to as the“above-ground” or “aerial” part, also often referred to as the “shoots”,and the “below-ground” part, also often referred to as the “roots”.

As used herein when discussing plants, the term “ovule” refers to thefemale gametophyte, whereas the term “pollen” means the malegametophyte.

As used herein, the term “plant tissue” refers to any part of a plant.Examples of plant organs include, but are not limited to the leaf, stem,root, tuber, seed, branch, pubescence, nodule, leaf axil, flower,pollen, stamen, pistil, petal, peduncle, stalk, stigma, style, bract,fruit, trunk, carpel, sepal, anther, ovule, pedicel, needle, cone,rhizome, stolon, shoot, pericarp, endosperm, placenta, berry, stamen,and leaf sheath.

As used herein, the term “phenotype” refers to the observable charactersof an individual cell, cell culture, organism (e.g., a plant), or groupof organisms which results from the interaction between thatindividual's genetic makeup (i.e., genotype) and the environment.

As used herein, the term “nucleic acid” refers to a polymeric form ofnucleotides of any length, either ribonucleotides ordeoxyribonucleotides, or analogs thereof. This term refers to theprimary structure of the molecule, and thus includes double- andsingle-stranded DNA, as well as double- and single-stranded RNA. It alsoincludes modified nucleic acids such as methylated and/or capped nucleicacids, nucleic acids containing modified bases, backbone modifications,and the like. The terms “nucleic acid” and “nucleotide sequence” areused interchangeably.

As used herein, the term “nucleotide change” or “nucleotidemodification” refers to, e.g., nucleotide substitution, deletion, and/orinsertion, as is well understood in the art. For example, suchnucleotide changes/modifications include mutations containingalterations that produce silent substitutions, additions, or deletions,but do not alter the properties or activities of the encoded protein orhow the proteins are made. As another example, such nucleotidechanges/modifications include mutations containing alterations thatproduce replacement substitutions, additions, or deletions, that alterthe properties or activities of the encoded protein or how the proteinsare made.

As used herein, the terms “polypeptide,” “peptide,” and “protein” areused interchangeably herein to refer to polymers of amino acids of anylength. These terms also include proteins that are post-translationallymodified through reactions that include glycosylation, acetylation andphosphorylation.

As used herein, the term “protein modification” refers to, e.g., aminoacid substitution, amino acid modification, deletion, and/or insertion,as is well understood in the art.

As used herein, the term “derived from” refers to the origin or source,and may include naturally occurring, recombinant, unpurified or purifiedmolecules. A nucleic acid or an amino acid derived from an origin orsource may have all kinds of nucleotide changes or protein modificationas defined elsewhere herein.

As used herein, the term “pathogen” refers to an agent that causesdisease, especially a living microorganism such as an insect, abacterium, virus, nematode or fungus.

As used herein, the term “nematode” refers to any of several worms ofthe phylum Nematoda, having unsegmented, cylindrical bodies, oftennarrowing at each end, and including parasitic forms such as thehookworm and pinworm. They are referred to as “roundworms” in somecontexts. Nematode species are very difficult to distinguish, with over28,000 having been described. Plant-parasitic nematodes include severalgroups causing severe crop losses. The most common genera areAphelenchoides (foliar nematodes), Ditylenchus, Globodera (potato cystnematodes), Heterodera (soybean cyst nematodes), Longidorus, Meloidogyne(root-knot nematodes), Nacobbus, Pratylenchus (lesion nematodes),Trichodorus and Xiphinema (dagger nematodes). Several phytoparasiticnematode species cause histological damages to roots, including theformation of visible galls (e.g. by root-knot nematodes), which areuseful characters for their diagnostic in the field. Some nematodespecies transmit plant viruses through their feeding activity on roots.One of them is Xiphinema index, vector of grapevine fanleaf virus), animportant disease of grapes. Other nematodes attacks bark and foresttrees. The most important representative of this group isBursaphelenchus xylophilus, the pine wood nematode, present in Asia andAmerica and recently discovered in Europe.

As used herein, the phrase “soybean cyst nematode” and the term “SCN”each refer to Heterodera glycines, a plant-parasitic nematode and adevastating pest of the soybean including Glycine max, worldwide. Thenematode infects the roots of soybean, and the female nematodeeventually becomes a cyst. Infection causes various symptoms that mayinclude chlorosis of the leaves: and stems, root necrosis, loss in seedyield and suppression of root and shoot growth. SCN has threatened theU.S. crop since the 1950s, reducing returns to soybean producers by $500million each year and reducing yields by as much as 75 percent. It isalso a significant problem in the soybean growing areas of the world,including South America and Asia.

As used herein, the term “resistant”, or “resistance”, describes aplant, line or cultivar that shows fewer or reduced symptoms to a bioticpest or pathogen than a susceptible (or more susceptible) plant, line orvariety to that biotic pest or pathogen. These terms are variouslyapplied to describe plants that show no symptoms as well as plantsshowing some symptoms but that are still able to produce marketableproduct with an acceptable yield. Some lines that are referred to asresistant are only so in the sense that they may still produce a crop,even though the plants may appear visually stunted and the yield isreduced compared to uninfected plants. As defined by the InternationalSeed Federation (ISF), a non-governmental, non-profit organizationrepresenting the seed industry (see “Definition of the Terms Describingthe Reaction of Plants to Pests or Pathogens and to Abiotic Stresses forthe Vegetable Seed Industry”, May 2005), the recognition of whether aplant is affected by or subject to a pest or pathogen can depend on theanalytical method employed. Resistance is defined by the ISF as theability of plant types to restrict the growth and development of aspecified pest or pathogen anchor the damage they cause when compared tosusceptible plant varieties under similar environmental conditions andpest or pathogen pressure. Resistant plant types may still exhibit somedisease symptoms or damage. Two levels of resistance are defined. Theterm “high/standard resistance” is used for plant varieties that highlyrestrict the growth and development of the specified pest or pathogenunder normal pest or pathogen pressure when compared to susceptiblevarieties. “Moderate/intermediate resistance” is applied to plant typesthat restrict the growth and development of the specified pest orpathogen, but exhibit a greater range of symptoms or damage compared toplant types with high resistance. Plant types with intermediateresistance will show less severe symptoms than susceptible plantvarieties, when grown under similar field conditions and pathogenpressure. Methods of evaluating resistance are well known to one skilledin the art. Such evaluation may be performed by visual observation of aplant or a plant part (e.g. leaves, roots, flowers, fruits et. al) indetermining the severity of symptoms. For example, when each plant isgiven a resistance score on a scale of 1 to 5 based on the severity ofthe reaction or symptoms, with 1 being the resistance score applied tothe most resistant plants (e.g., no symptoms, or with the leastsymptoms), and 5 the score applied to the plants with the most severesymptoms, then a line is rated as being resistant when at least 7.5% ofthe plants have a resistance score at a 1, 2, or 3 level, whilesusceptible lines are those having more than 25% of the plants scoringat a 4 or 5 level. If a more detailed visual evaluation is possible,then one can use a scale from 1 to 10 so as to broaden out the range ofscores and thereby hopefully provide a greater scoring spread among theplants being evaluated.

Another scoring system is a root inoculation test based on thedevelopment of the necrosis after inoculation and its position towardsthe cotyledon (such as one derived from Bosland et al., 1991), wherein 0stands for no symptom after infection; 1 stands for a small necrosis atthe hypocotyl after infection; 2 stands a necrosis under the cotyledonsafter infection; 3 stands for necrosis above the cotyledons afterinfection; 4 stands for a necrosis above the cotyledons together with awilt of the plant after infection, while eventually, 5 stands for a deadplant.

In addition to such visual evaluations, disease evaluations can beperformed by determining the pathogen bio-density in a plant or plantpart using electron microscopy and/or through molecular biologicalmethods, such as protein hybridization (e.g., ELISA, measuring pathogenprotein density) and/or nucleic acid hybridization (e.g., RT-PCR,measuring pathogen RNA density). Depending on the particularpathogen/plant combination, a plant may be determined resistant to thepathogen, for example, if it has a pathogen RNA/DNA and/or proteindensity that is about 50%, or about 40% or about 30%, or about 20%, orabout 10%, or about 5%, or about 2%, or about 1%, or about 0.1%, orabout 0.01%, or about 0.001%, or about 0.0001% of the RNA/DNA and/orprotein density in a susceptible plant.

Methods used in breeding plants for disease resistance are similar tothose used in breeding for other characters. It is necessary to know asmuch as possible about the nature of inheritance of the resistantcharacters in the host plant and the existence of physiological races orstrains of the pathogen.

As used herein, the term “fall resistance” is referred to as completefailure of the pathogen to develop after infection, and may either bethe result of failure of the pathogen to enter the cell (no initialinfection) or may be the result of failure of the pathogen to multiplyin the cell and infect subsequent cells (no subliminal infection, nospread). The presence of full resistance may be determined byestablishing the absence of pathogen protein or pathogen RNA in cells ofthe plant, as well as the absence of any disease symptoms in said plant,upon exposure of said plant to an infective dosage of pathogen (i.e.after ‘infection’). Among breeders, this phenotype is often referred toas “immune”. “Immunity” as used herein thus refers to a form ofresistance characterized by absence of pathogen replication even whenthe pathogen is actively transferred into cells by e.g. electroporation.

As used herein, the term “partial resistance” is referred to as reducedmultiplication of the pathogen in the cell, as reduced (systemic)movement of the pathogen, and/or as reduced symptom development afterinfection. The presence of partial resistance may be determined byestablishing the systemic presence of low concentration of pathogenprotein or pathogen RNA in the plant and the presence of decreased ordelayed disease-symptoms in said plant upon exposure of said plant to aninfective dosage of pathogen. Protein concentration may be determined byusing a quantitative detection method (e.g. an ELISA method or aquantitative reverse transcriptase-polymerase chain reaction (RT-PCR)).Among breeders, this phenotype is often referred to as “intermediateresistant.”

As used herein, the term “tolerant” is used herein to indicate aphenotype of a plant wherein disease-symptoms remain absent uponexposure of said plant to an infective dosage of pathogen, whereby thepresence of a systemic or local pathogen infection, pathogenmultiplication, at least the presence of pathogen genomic sequences incells of said plant and/or genomic integration thereof can beestablished. Tolerant plants are therefore resistant for symptomexpression but symptomless carriers of the pathogen. Sometimes, pathogensequences may be present or even multiply in plants without causingdisease symptoms. This phenomenon is also known as “latent infection”.In latent infections, the pathogen may exist in a truly latentnon-infectious occult form, possibly as an integrated genome or anepisomal agent (so that pathogen protein cannot be found in thecytoplasm, while PCR protocols may indicate the present of pathogennucleic acid sequences) or as an infectious and continuously replicatingagent. A reactivated pathogen may spread and initiate an epidemic amongsusceptible contacts. The presence of a “latent infection” isindistinguishable from the presence of a “tolerant” phenotype in aplant.

As used herein, the term “susceptible” is used herein to refer to aplant having no or virtually no resistance to the pathogen resulting inentry of the pathogen into the plant and multiplication and systemicspread of the pathogen, resulting in disease symptoms. The term“susceptible” is therefore equivalent to “non-resistant”.

As used herein, the term “offspring” refers to any plant resulting asprogeny from a vegetative or sexual reproduction from one or more parentplants or descendants thereof. For instance an offspring plant may beobtained by cloning or selfing of a parent plant or by crossing twoparents plants and include selfings as well as the F1 or F2 or stillfurther generations. An F1 is a first-generation offspring produced fromparents at least one of which is used for the first time as donor of atrait, while offspring of second generation (F2) or subsequentgenerations (F3, F4, etc.) are specimens produced from selfings of F1's,F2's etc. An F1 may thus be (and usually is a hybrid resulting from across between two true breeding parents (true-breeding is homozygous fora trait), while an F2 may be (and usually is an offspring resulting fromself-pollination of said F1 hybrids.

As used herein, the term “cross”, “crossing”, “cross pollination” or“cross-breeding” refer to the process by which the pollen of one floweron one plant is applied (artificially or naturally) to the ovule(stigma) of a flower on another plant.

As used herein the term “cultivar” refers to a variety, strain or raceof plant that has been produced by horticultural or agronomic techniquesand is not normally found in wild populations.

As used herein, the terms “dicotyledon,” “dicot” and “dicotyledonous”refer to a flowering plant having an embryo containing two seed halvesor cotyledons. Examples include tobacco; tomato; the legumes, includingpeas, alfalfa, clover and soybeans; oaks; maples; roses; mints;squashes; daisies; walnuts; cacti; violets and buttercups.

As used herein, the term “monocotyledon,” “monocot” or“monocotyledonous” refer to any of a subclass (Monocotyledoneae) offlowering plants having an embryo containing only one seed leaf andusually having parallel-veined leaves, flower parts in multiples ofthree, and no secondary growth in stems and roots. Examples includelilies; orchids; rice; corn, grasses, such as tall fescue, goat gas's,and Kentucky bluegrass; grains, such as wheat, oats and barley; irises;onions and palms.

As used herein, the term “gene” refers to any segment of DNA associatedwith a biological function. Thus, genes include, but are not limited to,coding sequences and/or the regulatory sequences required for theirexpression. Genes can also include nonexpressed DNA segments that, forexample, form recognition sequences for other proteins. Genes can beobtained from a variety of sources, including cloning from a source ofinterest or synthesizing from known or predicted sequence information,and may include sequences designed to have desired parameters.

As used herein, the term “genotype” refers to the genetic makeup of anindividual cell, cell culture, tissue, organism (e.g., a plant), orgroup of organisms.

As used herein, the term “allele(s)” means any of one or morealternative forms of a gene, all of which alleles relate to at least onetrait or characteristic. In a diploid cell, the two alleles of a givengene occupy corresponding loci on a pair of homologous chromosomes.Since the present invention relates, to QTLs, i.e. genomic regions thatmay comprise one or more genes or regulatory sequences, it is in someinstances more accurate to refer to “haplotype” (i.e. an allele of achromosomal segment) instead of “allele”, however, in those instances,the term “allele” should be understood to comprise the term “haplotype”.Alleles are considered identical when they express a similar phenotype.Differences in sequence are possible but not important as long as theydo not influence phenotype.

As used herein, the term “locus” (plural: “loci”) refers to any sitethat has been defined genetically. A locus may be a gene, or part of agene, or a DNA sequence that has some regulatory role, and may beoccupied by different sequences.

As used herein, the term “molecular marker” or “genetic marker” refersto an indicator that is used in methods for visualizing differences incharacteristics of nucleic acid sequences. Examples of such indicatorsare restriction fragment length polymorphism (RFLP) markers, amplifiedfragment length polymorphism (AFLP) markers, single nucleotidepolymorphisms (SNPs), insertion mutations, microsatellite markers(SSRs), sequence-characterized amplified regions (SCARs), cleavedamplified polymorphic sequence (CAPS) markers or isozyme markers orcombinations of the markers described herein which defines a specificgenetic and chromosomal location. Mapping of molecular markers in thevicinity of an allele is a procedure which can be performed quite easilyby the average person skilled in molecular-biological techniques whichtechniques are for instance described in Lefebvre and Chevre, 1995;Lorez and Wenzel, 2007, Srivastava and Narula, 2004, Meksem and Kahl,2005, Phillips and Vasil, 2001. General information concerning AFLPtechnology can be found in Vos et al. (1995, AFLP: a new technique forDNA fingerprinting, Nucleic Acids Res. 1995 Nov. 11; 23(21): 4107-1414).

As used herein, the term “hemizygous” refers to a cell, tissue ororganism in which a gene is present only once in a genotype, as a genein a haploid cell or organism, a sex-linked gene in the heterogameticsex, or a gene in a segment of chromosome in a diploid cell or organismwhere its partner segment has been deleted.

As used herein, the term “heterozygote” refers to a diploid or polyploidindividual cell or plant having different alleles (forms of a givengene) present at least at one locus. As used herein, the term“heterozygous” refers to the presence of different alleles (forms of agiven gene) at a particular gene locus.

As used herein, the term “homozygote” refers to an individual cell orplant having the same alleles at one or more loci.

As used herein, the term “homozygous” refers to the presence ofidentical alleles at one or more loci in homologous chromosomalsegments.

As used herein, the term “homologous” or “homolog” is known in the artand refers to related sequences that share a common ancestor or familymember and are determined based on the degree of sequence identity. Theterms “homology”, “homologous”, “substantially similar” and“corresponding substantially” are used interchangeably herein. Homologsusually control, mediate, or influence the same or similar biochemicalpathways, yet particular homologs may give rise to differing phenotypes.It is therefore understood, as those skilled in the art will appreciate,that the invention encompasses more than the specific exemplarysequences. These terms describe the relationship between a gene found inone species, subspecies, variety, cultivar or strain and thecorresponding or equivalent gene in another species, subspecies,variety, cultivar or strain. For purposes of this invention homologoussequences are compared.

The term “homolog” is sometimes used to apply to the relationshipbetween genes separated by the event of speciation (see “ortholog”) orto the relationship between genes separated by the event of geneticduplication (see “paralog”).

The term “ortholog” refers to genes in different species that evolvedfrom a common ancestral gene by speciation. Normally, orthologs retainthe same function in the course of evolution. Identification oforthologs is critical for reliable prediction of gene function in newlysequenced genomes.

The term “paralog” refers to genes related by duplication within agenome. While orthologs generally retain the same function in the courseof evolution, paralogs can evolve new functions, even if these arerelated to the original one.

“Homologous sequences” or “homologs” or “orthologs” are thought,believed, or blown to be functionally related. A functional relationshipmay be indicated in any one of a number of ways, including, but notlimited to (a) degree of sequence identity and/or (b) the same orsimilar biological function. Preferably, both (a) and (b) are indicated.The degree of sequence identity may vary, but in one embodiment, is atleast 50% (when using standard sequence alignment programs known in theart), at least 60%, at least 65%, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least about 91%, at least about 92%,at least about 93%, at least about 94%, at least about 95%, at leastabout 96%, at least about 97%, at least about 98%, or at least 98.5%, orat least about 99%, or at least 99.5%, or at least 99.8%, or at least99.9%. Homology can be determined using software programs readilyavailable in the art, such as those discussed in Current Protocols inMolecular Biology (F. M. Ausubel et al, eds., 1987) Supplement 30,section 7.718, Table 7.71. Some alignment programs are MacVector (OxfordMolecular Ltd, Oxford, U.K.) and ALIGN Plus (Scientific and EducationalSoftware, Pennsylvania). Other non-limiting alignment programs includeSequencher (Gene Codes, Ann Arbor, Mich.), AlignX, and Vector NTI(Invitrogen, Carlsbad, Calif.).

As used herein, the term “hybrid” refers to any individual cell, tissueor plant resulting from a cross between parents that differ in one ormore genes.

As used herein, the term “inbred” or “inbred line” refers to arelatively true-breeding strain.

The term “single allele converted plant” as used herein refers to thoseplants which are developed by a plant breeding technique calledbackcrossing wherein essentially all of the desired morphological andphysiological characteristics of an inbred are recovered in addition tothe single allele transferred into the inbred via the backcrossingtechnique.

As used herein, the term “line” is used broadly to include, but is notlimited to, a group of plants vegetatively propagated from a singleparent plant, via tissue culture techniques or a group of inbred plantswhich are genetically very similar due to descent from a commonparent(s). A plant is said to “belong” to a particular line if it (a) isa primary transformant (T0) plant regenerated from material of thatline: (b) has a pedigree comprised of a T0 plant of that line: or (c) isgenetically very similar due to common ancestry (e.g., via inbreeding orselfing). In this context, the term “pedigree” denotes the lineage of aplant, e.g. in terms of the sexual crosses affected such that a gene ora combination of genes, in heterozygous (hemizygous) or homozygouscondition, imparts a desired trait to the plant.

As used herein, the terms “introgression”, “introgressed” and“introgressing” refer to the process whereby genes of one species,variety or cultivar are moved into the genome of another species,variety or cultivar, by crossing those species. The crossing may benatural or artificial. The process may optionally be completed bybackcrossing to the recurrent parent, in which case introgression refersto infiltration of the genes of one species into the gene pool ofanother through repeated backcrossing of an interspecific hybrid withone of its parents. An introgression may also be described as aheterologous genetic material stably integrated in the genome of arecipient plant.

As used herein, the term “population” means a genetically homogeneous orheterogeneous collection of plants sharing a common genetic derivation.

As used herein, the term “variety” or “cultivar” means a group ofsimilar plants that by structural features and performance can beidentified from other varieties within the same species. The term“variety” as used herein has identical meaning to the correspondingdefinition in the International Convention for the Protection of NewVarieties of Plants (UPOV treaty), of Dec. 2, 1961, as Revised at Genevaon Nov. 10, 1972, on Oct. 23, 1978, and on Mar. 19, 1991. Thus,“variety” means a plant grouping within a single botanical taxon of thelowest known rank, which grouping, irrespective of whether theconditions for the grant of a breeder's right are fully met, can be i)defined by the expression of the characteristics resulting from a givengenotype or combination of genotypes, ii) distinguished from any otherplant grouping by the expression of at least one of the saidcharacteristics and iii) considered as a unit with regard to itssuitability for being propagated unchanged.

As used herein, the term “mass selection” refers to a form of selectionin which individual plants are selected and the next generationpropagated from the aggregate of their seeds. More details of massselection are described herein in the specification.

As used herein, the term “open pollination” refers to a plant populationthat is freely exposed to some gene flow, as opposed to a closed one inwhich there is an effective barrier to gene flow.

As used herein, the terms “open-pollinated population” or“open-pollinated variety” refer to plants normally capable of at leastsome cross-fertilization, selected to a standard, that may showvariation but that also have one or more genotypic or phenotypiccharacteristics by which the population or the variety can bedifferentiated from others. A hybrid, which has no barriers tocross-pollination, is an open-pollinated population or anopen-pollinated variety.

As used herein, the term “self-crossing”, “self pollinated” or“self-pollination” means the pollen of one flower on one plant isapplied (artificially or naturally) to the ovule (stigma) of the same ora different flower on the same plant.

As used herein, the term “cross”, “crossing”, “cross pollination” or“cross-breeding” refer to the process by which the pollen of one floweron one plant is applied (artificially or naturally) to the ovule(stigma) of a flower on another plant.

As used herein, the term “derived from” refers to the origin or source,and may include naturally occurring, recombinant, unpurified, orpurified molecules. A nucleic acid or an amino acid derived from anorigin or source may have all kinds of nucleotide changes or proteinmodification as defined elsewhere herein.

As used herein, the term “at least a portion” of a nucleic acid orpolypeptide means a portion having the minimal size characteristics ofsuch sequences, or any larger fragment of the full length molecule, upto and including the full length molecule. For example, a portion of anucleic acid may be 12 nucleotides. 13 nucleotides, 14 nucleotides, 15nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19nucleotides. 20 nucleotides, 22 nucleotides, 24 nucleotides, 26nucleotides, 28 nucleotides, 30 nucleotides, 32 nucleotides. 34nucleotides, 36 nucleotides, 38 nucleotides, 40 nucleotides, 45nucleotides, 50 nucleotides, 55 nucleotides, and so on, going up to thefull length nucleic acid. Similarly, a portion of a polypeptide may be 4amino acids, 5 amino acids, 6 amino acids, 7 amino acids, and so on,going up to the full length polypeptide. The length of the portion to beused will depend on the particular application. A portion of a nucleicacid useful as hybridization probe may be as short as 12 nucleotides; inone embodiment, it is 20 nucleotides. A portion of a polypeptide usefulas an epitope may be as short as 4 amino acids. A portion of apolypeptide that performs the function of the full-length polypeptidewould generally be longer than 4 amino acids.

As used herein, “sequence identity” or “identity” in the context of twonucleic acid or polypeptide sequences includes reference to the residuesin the two sequences which are the same when aligned for maximumcorrespondence over a specified comparison window. When percentage ofsequence identity is used in reference to proteins it is recognized thatresidue positions which are not identical often differ by conservativeamino acid substitutions, where amino acid residues are substituted forother amino acid residues with similar chemical properties (e.g., chargeor hydrophobicity) and therefore do not change the functional propertiesof the molecule. Where sequences differ in conservative substitutions,the percent sequence identity may be adjusted upwards to correct for theconservative nature of the substitution. Sequences which differ by suchconservative substitutions are said to have “sequence similarity” or“similarity.” Means for making this adjustment are well-known to thoseof skill in the art. Typically this involves scoring a conservativesubstitution as a partial rather than a full mismatch, therebyincreasing the percentage sequence identity. Thus, for example, where anidentical amino acid is given a score of 1 and a non-conservativesubstitution is given a score of zero, a conservative substitution isgiven a score between zero and 1. The scoring of conservativesubstitutions is calculated. e.g., according to the algorithm of Meyersand Miller, Computer Applic. Biol. Sci., 4:11-17 (1988).

As used herein, the term “suppression” or “disruption” of regulationrefers to reduced activity of regulatory proteins, and such reducedactivity can be achieved by a variety of mechanisms including antisense,mutation knockout or RNAi. Antisense RNA will reduce the level ofexpressed protein resulting in reduced protein activity as compared towild type activity levels. A mutation in the gene encoding a protein mayreduce the level of expressed protein and/or interfere with the functionof expressed protein to cause reduced protein activity.

As used herein, the terms “polynucleotide”, “polynucleotide sequence”,“nucleic acid sequence”, “nucleic acid fragment”, and “isolated nucleicacid fragment” are used interchangeably herein. These terms encompassnucleotide sequences and the like. A polynucleotide may be a polymer ofRNA or DNA that is single- or double-stranded, that optionally containssynthetic, non-natural or altered nucleotide bases. A polynucleotide inthe form of a polymer of DNA may be comprised of one or more segments ofcDNA, genomic DNA, synthetic DNA, or mixtures thereof. Nucleotides(usually found in their 5′-monophosphate form) are referred to by asingle letter designation as follows: “A” for adenylate ordeoxyadenylate (for RNA or DNA, respectively), “C” for cytidylate ordeoxycytidylate, “G” for guanylate or deoxyguanylate, “U” for uridylate,“T” for deoxythymidylate, “R” for purines (A or G), “Y” for pyrimidines(C or T), “K” for G or T, “H” for A or C or T, “I” for inosine, “W” forA or T, “S” for G or C, and “N” for any nucleotide.

Messenger RNAs (mRNAs) are gene-coding polynucleotide sequencestraditionally represented as strings of letters to marking the locationof each of the ribonucleic acids: “A” for adenylate, “C” for cytidylate,“G” for guanylate, and “U” for uridylate. Sequences for miRNA genes areusually written in the 5′monophosphate to 3′ hydroxyl orientation, andcan also be represented by their complementary DNA (cDNA) sequence byreplacing the “U” uridylates ribonucleotides with “T” deoxythymidylates.An example of this interchangeability between cDNA and mRNA sequences isillustrated in this application in the comparison of the cDNA sequenceof EG261 from Glycine pescadrensis (SEQ ID No. 5), and its respectivemRNA sequence (SEQ ID No: 66).

As used herein, “coding sequence” refers to a DNA sequence that codesfor a specific amino acid sequence. “Regulatory sequences” refer tonucleotide sequences located upstream (5′ non-coding sequences), within,or downstream (3′ non-coding sequences) of a coding sequence, and whichinfluence the transcription, RNA processing or stability, or translationof the associated coding sequence.

As used herein, “regulatory sequences” may include, but are not limitedto, promoters, translation leader sequences, introns, andpolyadenylation recognition sequences.

As used herein, “promoter” refers to a DNA sequence capable ofcontrolling the expression of a coding sequence or functional RNA. Thepromoter sequence consists of proximal and more distal upstreamelements, the latter elements often referred to as enhancers.Accordingly, an “enhancer” is a DNA sequence that can stimulate promoteractivity, and may be an innate element of the promoter or a heterologouselement inserted to enhance the level or tissue-specificity of apromoter. Promoters may be derived in their entirety from a native gene,or be composed of different elements derived from different promotersfound in nature, or even comprise synthetic DNA segments. It isunderstood by those skilled in the art that different promoters maydirect the expression of a gene in different tissues or cell types, orat different stages of development, or in response to differentenvironmental conditions. It is further recognized that since in mostcases the exact boundaries of regulatory sequences have not beencompletely defined, DNA fragments of some variation may have identicalpromoter activity. Promoters that cause a gene to be expressed in mostcell types at most times are commonly referred to as “constitutivepromoters”.

In some embodiments, the invention uses a Gmubi promoter to expressEG261 genes in root tissue. In other embodiments, alternative promotersare utilized including, but not limited to the: Cauliflower mosaic virus(CaMV) 35S promoter and enhanced 35S (E35S) promoter, the Cassava veinmosaic virus (CsVMV) promoter, the Figwort mosaic virus (FMV) promoter,and the Strawberry vein banding virus (SVBV2) promoter (see alsoHernandez-Garcia et al., 2010 (High level transgenic expression ofsoybean (Glycine max) GmERF and Gmubi gene promoters isolated by a novelpromoter analysis pipeline. BMC Plant Biol. 2010 Nov. 4; 10:237), andTucker et al., 2011 (Gene Expression Profiling and Shared Promoter Motiffor Cell Wall-Modifying Proteins Expressed in Soybean CystNematode-Infected Roots. Plant Phys. Vol 156 pp 319-329).

As used herein, the “3′ non-coding sequences” or “3′ UTR (untranslatedregion) sequence” refer to DNA sequences located downstream of a codingsequence and include polyadenylation recognition sequences and othersequences encoding regulatory signals capable of affecting mRNAprocessing or gene expression. The polyadenylation signal is usuallycharacterized by affecting the addition of polyadenylic acid tracts tothe 3′ end of the mRNA precursor. The use of different 3′ non-codingsequences is exemplified by Ingelbrecht, I. L., et al. (1989) Plant Cell1:671-680.

As used herein, the term “operably linked” refers to the association ofnucleic acid sequences on a single nucleic acid fragment so that thefunction of one is regulated by the other. For example, a promoter isoperably linked with a coding sequence when it is capable of regulatingthe expression of that coding sequence (i.e., that the coding sequenceis under the transcriptional control of the promoter). Coding sequencescan be operably linked to regulatory sequences in a sense or antisenseorientation. In another example, the complementary RNA regions of theinvention can be operably linked, either directly or indirectly, 5′ tothe target mRNA, or 3′ to the target mRNA, or within the target mRNA, ora first complementary region is 5′ and its complement is 3′ to thetarget mRNA.

As used herein, the term “vector”, “plasmid”, or “construct” refersbroadly to any plasmid or virus encoding an exogenous nucleic acid. Theterm should also be construed to include non-plasmid and non-viralcompounds which facilitate transfer of nucleic acid into virions orcells, such as, for example, polylysine compounds and the like. Thevector may be a viral vector that is suitable as a delivery vehicle fordelivery of the nucleic acid, or mutant thereof, to a cell, or thevector may be a non-viral vector which is suitable for the same purpose.Examples of viral and non-viral vectors for delivery of DNA to cells andtissues are well known in the art and are described, for example, in Maet al. (1997, Proc. Natl. Acad. Sci. U.S.A. 94:12744-12746). Examples ofviral vectors include, but are not limited to, recombinant plantviruses. Non-limiting examples of plant viruses include, TMV-mediated(transient) transfection into tobacco (Tuipe, T-H et al (1993), J.Virology Meth, 42: 227-239), ssDNA genomes viruses (e.g., familyGeminiviridae), reverse transcribing viruses (e.g., familiesCaulimoviridae, Pseudoviridae, and Metaviridae), dsNRA viruses (e.g.,families Reoviridae and Partitiviridae), (−) ssRNA viruses (e.g.,families Rhabdoviridae and Bunyraviridae), (−) ssRNA viruses (e.g.,families Bromoviridae, Closteroviridae, Comoviridae, Luteoviridae,Potyviridae, Sequiviridae and Tombusviridae) and viroids (e.g., familiesPospiviroidae and Avsunviroidae). Detailed classification information ofplant viruses can be found in Fauquet et al (2008, “Geminivirus straindemarcation and nomenclature”. Archives of Virology 153:783-821,incorporated herein by reference in its entirety), and Khan et al.(Plant viruses as molecular pathogens; Publisher Routledge, 2002, ISBN1560228954, 9781560228950). Examples of non-viral vectors include, butare not limited to, liposomes, polyamine derivatives of DNA, and thelike.

The present invention provides an isolated nucleic acid sequencecomprising a sequence selected from the group consisting of EG261,homologs of EG261, orthologs of EG261, paralogs of EG261, and fragmentsand variations thereof. In one embodiment, the present inventionprovides an isolated polynucleotide encoding the dirigent proteinproduced by the nucleic acid sequence for EG261, comprising a nucleicacid sequence that shares at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98% at least 99%, at least 99.1%, at least 99.2%, at least 99.3%,at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least99.8%, or at least 99.9% identity to EG261.

Methods of alignment of sequences for comparison are well known in theart. Various programs and alignment algorithms are described in: Smithand Waterman (Adv. Appl. Math., 2:482, 1981): Needleman and Wunsch (J.Mol. Biol., 48:443, 1970); Pearson and Lipman (Proc. Natl. Acad. Sci.,85:2444, 1988); Higgins and Sharp (Gene, 73:237-44, 1988); Higgins andSharp (CABIOS, 5:151-53, 1989); Corpet et al. (Nuc. Acids Res.,16:10881-90, 1988); Huang et al. (Comp. Appls Biosci. 8:155-65, 1992);and Pearson et al. (Meth. Mol. Biol., 24:307-31, 1994). Altschul et al(Nature Genet., 6:119-29, 1994) presents a detailed consideration ofsequence alignment methods and homology calculations.

The present invention also provides a chimeric gene comprising theisolated nucleic acid sequence of any one of the polynucleotidesdescribed above operably linked to suitable regulatory sequences.

The present invention also provides a recombinant construct comprisingthe chimeric gene as described above. In one embodiment, saidrecombinant construct is a gene silencing construct, such as used inRNAi gene silencing.

The expression vectors of the present invention will preferably includeat least one selectable marker. Such markers include dihydrofolatereductase, G418 or neomycin resistance for eukaryotic cell culture andtetracycline, kanamycin or ampicillin resistance genes for culturing inE. coli and other bacteria.

The present invention also provides a transformed host cell comprisingthe chimeric gene as described above. In one embodiment, said host cellis selected from the group consisting of bacteria, yeasts, filamentousfungi, algae, animals, and plants.

These sequences allow the design of gene-specific primers and probes forEG261, homologs of EG261, orthologs of EG261, paralogs of EG261, andfragments and variations thereof.

The term “primer” as used herein refers to an oligonucleotide which iscapable of annealing to the amplification target allowing a DNApolymerase to attach, thereby serving as a point of initiation of DNAsynthesis when placed under conditions in which synthesis of primerextension product is induced, i.e., in the presence of nucleotides andan agent for polymerization such as DNA polymerase and at a suitabletemperature and pH. The (amplification) primer is preferably singlestranded for maximum efficiency in amplification. Preferably, the primeris an oligodeoxyribonucleotide. The primer must be sufficiently long toprime the synthesis of extension products in the presence of the agentfor polymerization. The exact lengths of the primers will depend on manyfactors, including temperature and composition (A/T en G/C content) ofprimer. A pair of bi-directional primers consists of one forward and onereverse primer as commonly used in the art of DNA amplification such asin PCR amplification.

A probe comprises an identifiable, isolated nucleic acid that recognizesa target nucleic acid sequence. A probe includes a nucleic acid that isattached to an addressable location, a detectable label or otherreporter molecule and that hybridizes to a target sequence. Typicallabels include radioactive isotopes, enzyme substrates, co-factors,ligands, chemiluminescent or fluorescent agents, haptens, and enzymes.Methods for labelling and guidance in the choice of labels appropriatefor various purposes are discussed, for example, in Sambrook et al.(ed.), Molecular Cloning: A Laboratory Manual, 2^(nd) ed., vol. 1-3,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989 andAusubel e, al. Short Protocols in Molecular Biology, 4^(th) ed., JohnWiley & Sons, Inc., 1999.

Methods for preparing and using nucleic acid probes and primers aredescribed, for example, in Sambrook et al. (ed.). Molecular Cloning: ALaboratory Manual, 2^(nd) ed., vol. 1-3, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. 1989; Ausubel et al. Short Protocols inMolecular Biology, 4 ed., John Wiley & Sons, Inc., 1999; and Innis et alPCR Protocols, A Guide to Methods and Applications, Academic Press,Inc., San Diego, Calif., 1990. Amplification primer pairs can be derivedfrom a known sequence, for example, by using computer programs intendedfor that purpose such as PRIMER (Version 0.5, 1991, Whitehead Institutefor Biomedical Research, Cambridge, Mass.). One of ordinary skill in theart will appreciate that the specificity of a particular probe or primerincreases with its length. Thus, in order to obtain greater specificity,probes and primers can be selected that comprise at least 20, 25, 30,35, 40, 45, 50 or more consecutive nucleotides of a target nucleotidesequences.

As used herein, the term “soybean” refers to a plant of the Glycinegenus, including but not limited to the following species: G.clandestine, G. facata, G. latifolia, G. latrobeana, G. canescens, G.tabacina, G. tomentella, G. soja; G. max, G. Arenaria, G. argyrea, G.canescens, G. clandestine, G. curvata, G. cyrtoloba, G. microphylla, G.pescadrensis, G. pindanica, G. rubigiosa, G. soja, and G. Stenophita(see for example U.S. Pat. No. 8,389,798, and Verma et al., (1996)Soybean: genetics, molecular biology and biotechnology, Verma, D. P. S.Shoemaker, R. C. (editors)).

As used herein, the term “garden bean” or “bean” refers to a plant ofthe Phaseolus genus, including but not limited to the following species:P. acutifolius, P. aegpticus, P. amblyosepalus, P. angastissimus, P.coccineus, P. filiformis, P. lunatus, P. Maculates, and P. Vulgaris (seefor example Turley et al., 1999 (Phylogenetic Analysis of the Cultivatedand Wild Species of Phaseolus (Fabaceae), Systematic Botany Vol. 24, No.3 (July-September, 199) pp 438-460)).

As used herein, the term “cowpea” refers to a plant of the Vigna genus,including but not limited to the following species: V. aconitfolia, K.angularis, V. caracalla, V. lanceolota, V. o-wahuensis, V. umbellata, V.subterranea, and V. Unguiculata (see for example Sylla Ba et al., 2004(Genetic diversity in cowpea [Vigna unguiculata (L.) Walp.] as revealedby RAPD markers Genetic Resources and Crop Evolution 51: 539-550, 2004,and Pasquet et al., (2001) Cowpea. In: Tropical Plant Breeding. CharrierA., Jacquot M., Hamon S, and Nicolas D. (editors), Science publishers,Enfield, pp. 177-198.)).

EG261 Proteins

The present invention also provides polypeptides and amino acidsequences comprising at least a portion of the isolated proteins encodedby nucleotide sequences for EG261, homologs of EG261, orthologs ofEG261, paralogs of EG261, and fragments and variations thereof.

The present invention also provides an isolated amino acid sequenceencoded by the nucleic acid sequences of EG261, homologs of EG261,orthologs of EG261, paralogs of EG261, and/or fragments and variationsthereof. In some embodiments, the present invention provides an isolatedpolypeptide comprising an amino acid sequence that shares at least about90% about 91%, about 92%, about 93%, about 94%, about 95%, about 96%,about 97% about 98%, about 99%, about 99.1%, about 99.2%, about 99.3%,about 99.4%, about 99.5%, about 99.6%, about 99.7%, about 99.8%, orabout 99.9% identity to an amino acid sequence encoded by the nucleicacid sequences of EG261, homologs of EG261, orthologs of EG261, paralogsof EG261, and/or fragments and variations thereof. In one embodiment,the present invention provides an isolated polypeptide comprising anamino acid sequence which encodes an amino acid sequence that shares atleast about 85%, about 86%, about 87%, about 88% about 89%, about 90%,about 91%, about 92%, about 93%, about 94%, about 95% about 96%, about97%, about 98%, about 99%, about 99.1%, about 99.2%, about 99.3%, about99.4%, about 99.5%, about 99.6%, about 99.7%, about 99.8, or about 99.9%identity to an amino acid sequence encoded by the nucleic acid sequencesof EG261, homologs of EG261, orthologs of EG261, paralogs of EG261,and/or fragments and variations thereof.

The invention also encompasses variants and fragments of proteins of anamino acid sequence encoded by the nucleic acid sequences of EG261,homologs of EG261, orthologs of EG261 and/or paralogs of EG261. Thevariants may contain alterations in the amino acid sequences of theconstituent proteins. The term “variant” with respect to a polypeptiderefers to an amino acid sequence that is altered by one or more aminoacids with respect to a reference sequence. The variant can have“conservative” changes, or “nonconservative” changes, e.g., analogousminor variations can also include amino acid deletions or insertions, orboth.

Functional fragments and variants of a polypeptide include thosefragments and variants that maintain one or more functions of the parentpolypeptide. It is recognized that the gene or cDNA encoding apolypeptide can be considerably mutated without materially altering oneor more of the polypeptide's functions. First, the genetic code iswell-known to be degenerate, and thus different codons encode the sameamino acids. Second, even where an amino acid substitution isintroduced, the mutation can be conservative and have no material impacton the essential function(s) of a protein. See, e.g., StryerBiochemistry 3^(rd) Ed. 1988. Third, part of a polypeptide chain can bedeleted without impairing or eliminating all of its functions. Fourth,insertions or additions can be made in the polypeptide chain forexample, adding epitope tags, without impairing or eliminating itsfunctions (Ausubel et al. J. Immunol. 159(5): 2502-12, 1997). Othermodifications that can be made without materially impairing one or morefunctions of a polypeptide can include, for example, in vivo or in vitrochemical and biochemical modifications or the incorporation of unusualamino acids. Such modifications include, but are not limited to, forexample, acetylation, carboxylation, phosphorylation, glycosylation,ubiquination, labelling, e.g., with radionucleotides, and variousenzymatic modifications, as will be readily appreciated by those wellskilled in the art. A variety of methods for labelling polypeptides, andlabels useful for such purposes, are well known in the art, and includeradioactive isotopes such as ³²P, ligands which bind to or are bound bylabelled specific binding partners (e.g., antibodies), fluorophores,chemiluminescent agents, enzymes, and anti-ligands. Functional fragmentsand variants can be of varying length. For example, some fragments haveat least 10, 25, 50, 75, 100, 200, or even more amino acid residues.These mutations can be natural or purposely changed. In someembodiments, mutations containing alterations that produce silentsubstitutions, additions, or deletions, but do not alter the propertiesor activities of the proteins or how the proteins are made are anembodiment of the invention.

Conservative amino acid substitutions are those substitutions that, whenmade, least interfere with the properties of the original protein thatis, the structure and especially the function of the protein isconserved and not significantly changed by such substitutions.Conservative substitutions generally maintain (a) the structure of thepolypeptide backbone in the area of the substitution, for example, as asheet or helical conformation, (b) the charge or hydrophobicity of themolecule at the target site, or (c) the bulk of the side chain. Furtherinformation about conservative substitutions can be found, for instance,in Ben Bassat et al. (J. Bacteriol., 169:751-757, 1987), O'Regan et ac.(Gene, 77:237-251, 1989), Sahin-Toth et al. (Protein Sci., 3:240-247,1994). Hochuli et al. (Bio/Technology, 6:1321-1325, 1988) and in widelyused textbooks of genetics and molecular biology. The Blosun matricesare commonly used for determining the relatedness of polypeptidesequences. The Blosum matrices were created using a large database oftrusted alignments (the BLOCKS database), in which pairwise sequencealignments related by less than some threshold percentage identity werecounted (Henikoff et al., Proc. Natl. Acad. Sci. USA, 89:10915-10919,1992). A threshold of 90%6 identity was used for the highly conservedtarget frequencies of the BLOSUM90 matrix. A threshold of 65% identitywas used for the BLOSUM65 matrix. Scores of zero and above in the Blosunmatrices are considered “conservative substitutions” at the percentageidentity selected. The following table shows exemplary conservativeamino acid substitutions.

Very Highly - Highly Conserved Original Conserved Substitutions (fromthe Conserved Substitutions Residue Substitutions Blosum90 Matrix) (fromthe Blosum65 Matrix) Ala Ser Gly, Ser, Thr Cys, Gly, Ser, Thr, Val ArgLys Gln, His, Lys Asn, Gln, Glu, His, Lys Asn Gln; His Asp, Gln, His,Lys, Ser, Thr Arg, Asp, Gln, Glu, His, Lys, Ser, Thr Asp Glu Asn, GluAsn, Gln, Glu, Ser Cys Ser None Ala Gln Asn Arg, Asn, Glu, His, Lys, MetArg, Asn, Asp, Glu, His, Lys, Met, Ser Glu Asp Asp, Gln, Lys Arg, Asn,Asp, Gln, His, Lys, Ser Gly Pro Ala Ala, Ser His Asn; Gln Arg, Asn, Gln,Tyr Arg, Asn, Gln, Glu, Tyr Ile Leu; Val Leu, Met, Val Leu, Met, Phe,Val Leu Ile; Val Ile, Met, Phe, Val Ile, Met, Phe, Val Lys Arg; Gln; GluArg, Asn, Gln, Glu Arg, Asn, Gln, Glu, Ser, Met Leu; Ile Gln, Ile, Leu,Val Gln, Ile, Leu, Phe, Val Phe Met; Leu; Tyr Leu, Trp, Tyr Ile, Leu,Met, Trp, Tyr Ser Thr Ala, Asn, Thr Ala, Asn, Asp, Gln, Glu, Gly, Lys,Thr Thr Ser Ala, Asn, Ser Ala, Asn, Ser, Val Trp Tyr Phe, Tyr Phe, TyrTyr Trp; Phe His, Phe, Trp His, Phe, Trp Val Ile; Leu Ile, Leu, Met Ala,Ile, Leu, Met, Thr

In some examples, variants can have no more than 3, 5, 10, 15, 20, 25,30, 40, 50, or 100 conservative amino acid changes (such as very highlyconserved or highly conserved amino acid substitutions). In otherexamples, one or several hydrophobic residues (such as Len, Ile, Val,Met, Phe, or Trp) in a variant sequence can be replaced with a differenthydrophobic residue (such as Len, Ile, Val. Met, Phe, or Tip) to createa variant functionally similar to the disclosed an amino acid sequencesencoded by the nucleic acid sequences of EG261, homologs of EG261,orthologs of EG261 and/or paralogs of EG261, and/or fragments andvariations thereof.

In some embodiments, variants may differ from the disclosed sequences byalteration of the coding region to fit the codon usage bias of theparticular organism into which the molecule is to be introduced. Inother embodiments, the coding region may be altered by taking advantageof the degeneracy of the genetic code to alter the coding sequence suchthat, while the nucleotide sequence is substantially altered, itnevertheless encodes a protein having an amino acid sequencesubstantially similar to the disclosed an amino acid sequences encodedby the nucleic acid sequences of EG261, homologs of EG261, orthologs ofEG261 and/or paralogs of EG261, and/or fragments and variations thereof.

In some embodiments, functional fragments derived from the EG261orthologs of the present invention are provided. The functionalfragments can still confer resistance to pathogens when expressed in aplant. In some embodiments, the functional fragments contain at leastthe dirigent domain of a wild type EG261 orthologs, or functionalvariants thereof. In some embodiments, the functional fragments containone or more conserved region shared by two or more EG261 orthologs,shared by two or more EG261 orthologs in the same plant genus, shared bytwo or more dicot EG261 orthologs, and/or shared by two or more monocotEG261 orthologs. Non-limiting exemplary conserved regions are shown inFIGS. 2 to 4. The dirigent domain or conserved regions can be determinedby any suitable computer program, such as NCBI protein BLAST program andNCBI Alignment program, or equivalent programs. In some embodiments, thefunctional fragments are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50or more amino acids shorter compared to the EG261 orthologs of thepresent invention. In some embodiments, the functional fragments aremade by deleting one or more amino acid of the EG261 orthologs of thepresent invention. In some embodiments, the functional fragments shareat least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more identity to theEG261 orthologs of the present invention.

In some embodiments, functional chimeric or synthetic polypeptidesderived from the EG261 orthologs of the present invention are provided.The functional chimeric or synthetic polypeptides can still conferresistance to pathogens when expressed in a plant. In some embodiments,the functional chimeric or synthetic polypeptides contain at least thedirigent domain of a wild type EG261 orthologs, or functional variantsthereof. In some embodiments, the functional chimeric or syntheticpolypeptides contain one or more conserved region shared by two or moreEG261 orthologs, shared by two or more EG261 orthologs in the same plantgenus, shared by two or more dicot EG261 orthologs, and/or shared by twoor more monocot EG261 orthologs. Non-limiting exemplary conservedregions are shown in FIGS. 2 to 4. The dirigent domain or conservedregions can be determined by any suitable computer program, such as NCBIprotein BLAST program and NCBI Alignment program, or equivalentprograms. In some embodiments, the functional chimeric or syntheticpolypeptides share at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, ormore identity to the EG261 orthologs of the present invention.

Sequences of conserved regions can also be used to knock-down the levelof one or more EG261 orthologs. In some embodiments, sequences ofconserved regions can be used to make gene silencing molecules to targetone ore more EG261 orthologs. In some embodiments, the gene silencingmolecules are selected from the group consisting of double-strandedpolynucleotides, single-stranded polynucleotides or Mixed DuplexOligonucleotides. In some embodiments, the gene silencing moleculescomprises a DNA/RNA fragment of about 10 bp, 15 bp, 19 bp, 20 bp, 21 bp,25 bp, 30 bp, 40 bp, 50 bp, 60 bp, 70 bp, 80 bp, 90 bp, 100 bp, 150 bp,200 bp, 250 bp, 300 bp, 350 bp, 400 bp, 500 bp, 600 bp, 700 bp, 800 bp,900 bp, 1000 bp, or more polynucleotides, wherein the DNA/RNA fragmentshare at least 90%, 95%, 99%, or more identity to a conserved region ofthe EG261 orthologs sequences of the present invention, or complementarysequences thereof.

Plant Transformation

The present polynucleotides coding for EG261, homologs of EG261,orthologs of EG261 and/or paralogs of EG261, and/or fragments andvariations thereof of the present invention can be transformed intosoybean or other plant genera.

Methods of producing transgenic plants are well known to those ofordinary skill in the art. Transgenic plants can now be produced by avariety of different transformation methods including, but not limitedto, electroporation; microinjection; microprojectile bombardment, alsoknown as particle acceleration or biolistic bombardment: viral-mediatedtransformation; and Agrobacterium-mediated transformation. See, forexample, U.S. Pat. Nos. 5,405,765; 5,472,869; 5,538,877; 5,538,880;5,550,318; 5,641,664; 5,736,369 and 5,736,369; International PatentApplication Publication Nos. WO2002/038779 and WO/2009/117555: La etal., (Plant Cell Reports, 2008, 27:273-278): Watson et al., RecombinantDNA, Scientific American Books (1992); Hinchee et al., Bio/Tech.6:915-922 (1988); McCabe et al., Bio/Tech. 6:923-926 (1988); Toriyama etal., Bio/Tech. 6:1072-1074 (1988); Fromm et al., Bio/Tech. 8:833-839(1990) Mullins et al., Bio/Tech. 8:833-839 (1990); Hiei et al. PlantMolecular Biology 35:205-218 (1997): Ishida et al., Nature Biotechnology14:745-750 (1996); Zhang et al., Molecular Biotechnology 8:223-231(1997); Ku et al., Nature Biotechnology 17:76-80 (1999); and, Raineri etal., Bio/Tech. 8:33-38 (1990)), each of which is expressly incorporatedherein by reference in their entirely.

Agrobacterium tumefaciens is a naturally occurring bacterium that iscapable of inserting its DNA (genetic information) into plants,resulting in a type of injury to the plant known as crown gall. Mostspecies of plants can now be transformed using this method, includingcucurbitaceous species.

Microprojectile bombardment is also known as particle acceleration,biolistic bombardment, and the gene gun (Biolistic® Gene Gun). The genegun is used to shoot pellets that are coated with genes (e.g. fordesired traits) into plant seeds or plant tissues in order to get theplant cells to then express the new genes. The gene gum uses an actualexplosive (.22 caliber blank) to propel the material. Compressed air orsteam may also be used as the propellant. The Biolistic® Gene Gun wasinvented in 1983-1984 at Cornell University by John Sanford, EdwardWolf, and Nelson Allen. It and its registered trademark are now owned byE. I. du Pont de Nemours and Company. Most species of plants have beentransformed using this method.

The most common method for the introduction of new genetic material intoa plant genome involves the use of living cells of the bacterialpathogen Agrobacterium tumefaciens to literally inject a piece of DNA,called transfer or T-DNA, into individual plant cells (usually followingwounding of the tissue) where it is targeted to the plant nucleus forchromosomal integration. There are numerous patents governingAgrobacterium mediated transformation and particular DNA deliveryplasmids designed specifically for use with Agrobacterium—for example,U.S. Pat. No. 4,536,475, EP0265556, EP0270822, WO8504899, WO8603516,U.S. Pat. No. 5,591,616, EP0604662, EP0672752, WO8603776, WO9209696,WO09419930, WO9967357, U.S. Pat. No. 4,399,216, WO8303259, U.S. Pat. No.5,731,179. EP068730, WO09516031, U.S. Pat. No. 5,693,512, U.S. Pat. No.6,051,757 and EP904362A1. Agrobacterium-mediated plant transformationinvolves as a first step the placement of DNA fragments cloned onplasmids into living Agrobacterium cells, which are then subsequentlyused for transformation into individual plant cells.Agrobacterium-mediated plant transformation is thus an indirect planttransformation method. Methods of Agrobacterium-mediated planttransformation that involve using vectors with no T-DNA are also wellknown to those skilled in the art and can have applicability in thepresent invention. See, for example, U.S. Pat. No. 7,250,554, whichutilizes P-DNA instead of T-DNA in the transformation vector.

A transgenic plant formed using Agrobacterium transformation methodstypically contains a single gene on one chromosome, although multiplecopies are possible. Such transgenic plants can be referred to as beinghemizygous for the added gene. A more accurate name for such a plant isan independent segregant, because each transformed plant represents aunique T-DNA integration event (U.S. Pat. No. 6,156,953). A transgenelocus is generally characterized by the presence and/or absence of thetransgene. A heterozygous genotype in which one allele corresponds tothe absence of the transgene is also designated hemizygous (U.S. Pat.No. 6,008,437).

Direct plant transformation methods using DNA have also been reported.The first of these to be reported historically is electroporation, whichutilizes an electrical current applied to a solution containing plantcells (M. E. Fromm et al., Nature, 319, 791 (1986); H. Jones et al.,Plant Mol. Biol., 13, 501 (1989) and H. Yang et al., Plant Cell Reports,7, 421 (1988). Another direct method, called “biolistic bombardment”,uses ultrafine particles, usually tungsten or gold, that are coated withDNA and then sprayed onto the surface of a plant tissue with sufficientforce to cause the particles to penetrate plant cells, including thethick cell wall, membrane and nuclear envelope, but without killing atleast some of them (U.S. Pat. No. 5,204,253, U.S. Pat. No. 5,015,580). Athird direct method uses fibrous forms of metal or ceramic consisting ofsharp, porous or hollow needle-like projections that literally impalethe cells, and also the nuclear envelope of cells. Both silicon carbideand aluminum borate whiskers have been used for plant transformation(Mizmo et al., 2004; Petolino et al., 2000; U.S. Pat. No. 5,302,523 USApplication 20040197909) and also for bacterial and animaltransformation (Kaepler et al., 1992: Raloff, 1990; Wang, 1995). Thereare other methods reported, and undoubtedly, additional methods will bedeveloped. However, the efficiencies of each of these indirect or directmethods in introducing foreign DNA into plant cells are invariablyextremely low, making it necessary to use some method for selection ofonly those cells that have been transformed, and further, allowinggrowth and regeneration into plants of only those cells that have beentransformed.

For efficient plant transformation, a selection method must be employedsuch that whole plants are regenerated from a single transformed celland every cell of the transformed plant carries the DNA of interest.These methods can employ positive selection, whereby a foreign gene issupplied to a plant cell that allows it to utilize a substrate presentin the medium that it otherwise could not use, such as mannose or xylose(for example, refer U.S. Pat. No. 5,767,378; U.S. Pat. No. 5,994,629).More typically, however, negative selection is used because it is moreefficient, utilizing selective agents such as herbicides or antibioticsthat either kill or inhibit the growth of non-transformed plant cellsand reducing the possibility of chimeras.

Resistance genes that are effective against negative selective agentsare provided on the introduced foreign DNA used for the planttransformation. For example, one of the most popular selective agentsused is the antibiotic kanamycin, together with the resistance geneneomycin phosphotransferase (nptII), which confers resistance tokanamycin and related antibiotics (see, for example, Messing & Viena,Gene 19: 259-268 (1982); Bevan et al., Nature 304:184-187 (1983)).However, many different antibiotics and antibiotic resistance genes canbe used for transformation proposes (refer U.S. Pat. No. 5,034,322, U.S.Pat. No. 6,174,724 and U.S. Pat. No. 6,255,560). In addition, severalherbicides and herbicide resistance genes have been used fortransformation purposes, including the bar gene, which confersresistance to the herbicide phosphinothricin (White et al., Nucl AcidsRes 18: 1062 (1990), Spencer et al., Theor Appl Genet 79: 625-631(1990), U.S. Pat. No. 4,795,855, U.S. Pat. No. 5,378,824 and U.S. Pat.No. 6,107,549). In addition, the dhfr gene, which confers resistance tothe anticancer agent methotrexate, has been used for selection (Bourouiset al., EMBO J. 2(7): 1099-1104 (1983).

Non-limiting examples of binary vectors suitable for soybean speciestransformation and transformation methods are described by Yi et al.2006 (Transformation of multiple soybean cultivars by infectingcotyledonary-node with Agrobacterium tumefaciens, African Journal ofBiotechnology Vol. 5 (20), pp. 1989-1993, 16 Oct. 2006), Paz et al. 2004(Assessment of conditions affecting Agrobacterium-mediated soybeantransformation using the cotyledonary node explant. Euphytica 136:167-179, 2004), U.S. Pat. Nos. 5,376,543, 5,416,011, 5,968,830, and5,569,834, or by similar experimental procedures well known to thoseskilled in the art. Soybean plants can be transformed by using anymethod described in the above references.

The expression control elements used to regulate the expression of agiven protein can either be the expression control element that isnormally found associated with the coding sequence (homologousexpression element) or can be a heterologous expression control element.A variety of homologous and heterologous expression control elements areknown in the art and can readily be used to make expression units foruse in the present invention. Transcription initiation regions, forexample, can include any of the various opine initiation regions, suchas octopine, mannopine, nopaline and the like that are found in the Tiplasmids of Agrobacterium tumefaciens. Alternatively, plant viralpromoters can also be used, such as the cauliflower mosaic virus 19S and35S promoters (CaMV 19S and CaMV 35S promoters, respectively) to controlgene expression in a plant (U.S. Pat. Nos. 5,352,605; 5,530,196 and5,858,742 for example). Enhancer sequences derived from the CaMV canalso be utilized (U.S. Pat. Nos. 5,164,316; 5,196,525; 5,322,938;5,530,196; 5,352,605; 5,359,142; and 5,858,742 for example). Plantpromoters such as prolifera promoter, fruit specific promoters, Ap3promoter, heat shock promoters, seed specific promoters, etc. can alsobe used.

In some embodiments, EG261 genes can be expressed using promoters fromnative EG261 genes. Thus in some embodiments expression of any one ofthe EG261 variants of the present invention can be driven by said EG261gene's own native promoter. For example, in some embodiments, a geneencoding for the EG261 polypeptide from G. pescadrensis (SEQ ID NO: 12)may be expressed using the native G. pescarensis EG261 promoter (SEQ IDNO: 69). In other embodiments, a gene encoding for the EG261 polypeptidefrom G. pescadrensis (SEQ ID NO: 12) may be expressed using the nativeG. max EG261 promoter (SEQ ID NO: 70). In one embodiment, the EG261 geneoperably linked to the EG261 native promoter is transformed andexpressed in G. max.

Persons having skill in the art will also appreciate that in someembodiments, EG261 genes may also be expressed by the native promotersof other EG261 wild variants, homologs or orthologs. For example, a geneencoding for the EG261 polypeptide from G. tomatella from Australia (SEQID NO: 15) may be expressed using the native G. pescadrensis EG261promoter (SEQ ID NO: 69), or the G. max native promoter (SEQ ID NO: 70).Thus in some embodiments, the EG261 genes of the present invention maybe expressed using any of the native promoters of EG261 wild variants,homologs, or orthologs.

Either a gamete-specific promoter, a constitutive promoter (such as theCaMV or Nos promoter), an organ-specific promoter (such as the E8promoter from tomato), or an inducible promoter is typically ligated tothe protein or antisense encoding region using standard techniques knownin the art. The expression unit may be further optimized by employingsupplemental elements such as transcription terminators and/or enhancerelements.

Thus, for expression in plants, the expression units will typicallycontain, in addition to the protein sequence, a plant promoter region, atranscription initiation site and a transcription termination sequence.Unique restriction enzyme sites at the 5′ mid 3′ ends of the expressionunit are typically included to allow for easy insertion into apre-existing vector.

In the construction of heterologous promoter/structural gene orantisense combinations, the promoter is preferably positioned about thesame distance from the heterologous transcription start site as it isfrom the transcription start site in its natural setting. As is known inthe art, however, some variation in this distance can be accommodatedwithout loss of promoter function.

In addition to a promoter sequence, the expression cassette can alsocontain a transcription termination region downstream of the structuralgene to provide for efficient termination. The termination region may beobtained from the same gene as the promoter sequence or may be obtainedfrom different genes. If the mRNA encoded by the structural gene is tobe efficiently processed, DNA sequences which direct polyadenylation ofthe RNA are also commonly added to the vector construct. Polyadenylationsequences include, but are not limited to the Agrobacterium octopinesynthase signal (Gielen et al., EMBO J 3:835-846 (1984)) or the nopalinesynthase signal (Depicker et al., Mol. and Appl. Genet. 1:561-573(1982)). The resulting expression unit is ligated into or otherwiseconstructed to be included in a vector that is appropriate for higherplant transformation. One or more expression units may be included inthe same vector. The vector will typically contain a selectable markergene expression unit by which transformed plant cells can be identifiedin culture. Usually, the marker gene will encode resistance to anantibiotic, such as G418, hygromycin, bleomycin, kanamycin, orgentamicin or to an herbicide, such as glyphosate (Round-Up) orglufosinate (BASTA) or atrazine. Replication sequences, of bacterial orviral origin, are generally also included to allow the vector to becloned in a bacterial or phage host; preferably a broad host range forprokaryotic origin of replication is included. A selectable marker forbacteria may also be included to allow selection of bacterial cellsbearing the desired construct. Suitable prokaryotic selectable markersinclude resistance to antibiotics such as ampicillin, kanamycin ortetracycline. Other DNA sequences encoding additional functions may alsobe present in the vector, as is known in the art. For instance, in thecase of Agrobacterium transformations, T-DNA sequences will also beincluded for subsequent transfer to plant chromosomes.

To introduce a desired gene or set of genes by conventional methodsrequires a sexual cross between two lines, and then repeatedback-crossing between hybrid offspring and one of the parents until aplant with the desired characteristics is obtained. This process,however, is restricted to plants that can sexually hybridize, and genesin addition to the desired gene will be transferred.

Recombinant DNA techniques allow plant researchers to circumvent theselimitations by enabling plant geneticists to identify and clone specificgenes for desirable traits, such as improved fatty acid composition, andto introduce these genes into already useful varieties of plants. Oncethe foreign genes have been introduced into a plant, that plant can thenbe used in conventional plant breeding schemes (e.g., pedigree breeding,single-seed-descent breeding schemes, reciprocal recurrent selection) toproduce progeny which also contain the gene of interest.

Genes can be introduced in a site directed fashion using homologousrecombination. Homologous recombination permits site-specificmodifications in endogenous genes and thus inherited or acquiredmutations may be corrected, and/or novel alterations may be engineeredinto the genome. Homologous recombination and site-directed integrationin plants are discussed in, for example, U.S. Pat. Nos. 5,451,513;5,501,967 and 5,527,695.

Breeding Methods

Open-Pollinated Populations.

The improvement of open-pollinated populations of such crops as rye,many maizes and sugar beets, herbage grasses, legumes such as alfalfaand clover, and tropical tree crops such as cacao, coconuts, oil palmand some rubber, depends essentially upon changing gene-frequenciestowards fixation of favorable alleles while maintaining a high (but farfrom maximal) degree of heterozygosity. Uniformity in such populationsis impossible and trueness-to-type in an open-pollinated variety is astatistical feature of the population as a whole, not a characteristicof individual plants. Thus, the heterogeneity of open-pollinatedpopulations contrasts with the homogeneity (or virtually so) of inbredlines, clones and hybrids.

Population improvement methods fall naturally into two groups, thosebased on purely phenotypic selection, normally called mass selection,and those based on selection with progeny testing. Interpopulationimprovement utilizes the concept of open breeding populations; allowinggenes for flow from one population to another. Plants in one population(cultivar, strain, ecotype, or any germplasm source) are crossed eithernaturally (e.g. by wind) or by hand or by bees (commonly Apis melliferaL. or Megachile rotundata F.) with plants from other populations.Selection is applied to improve one (or sometimes both) population(s) byisolating plants with desirable traits from both sources.

There are basically two primary methods of open-pollinated populationimprovement. First, there is the situation in which a population ischanged en masse by a chosen selection procedure. The outcome is animproved population that is indefinitely propagable by random-matingwithin itself in isolation. Second, the synthetic variety attains thesame end result as population improvement but is not itself propagableas such; it has to be reconstructed from parental lines or clones. Theseplant breeding procedures for improving open-pollinated populations arewell known to those skilled in the art and comprehensive reviews ofbreeding procedures routinely used for improving cross-pollinated plantsare provided in numerous texts and articles, including: Allard,Principles of Plant Breeding, John Wiley & Sons, Inc. (1960); Simmonds,Principles of Crop Improvement, Longman Group Limited (1979); Hallauerand Miranda, Quantitative Genetics in Maize Breeding, Iowa StateUniversity Press (1981); and, Jensen, Plant Breeding Methodology, JohnWiley & Sons, Inc. (1988). For population improvement methods specificfor soybean see, e.g., J. R. Wilcox, editor (1987) SOYBEANS:Improvement, Production, and Uses, Second Edition, American Society ofAgronomy, Inc., Crop Science Society of America, Inc., and Soil ScienceSociety of America, Inc., publishers, 888 pages.

Mass Selection.

In mass selection, desirable individual plants are chosen, harvested,and the seed composited without progeny testing to produce the followinggeneration. Since selection is based on the maternal parent only, andthere is no control over pollination, mass selection amounts to a formof random mating with selection. As stated above, the purpose of massselection is to increase the proportion of superior genotypes in thepopulation.

Synthetics.

A synthetic variety is produced by crossing inter se a number ofgenotypes selected for good combining ability in all possible hybridcombinations, with subsequent maintenance of the variety by openpollination. Whether parents are (more or less inbred) seed-propagatedlines, as in some sugar beet and beans (Vicia) or clones, as in herbagegrasses, clovers and alfalfa, makes no difference in principle. Parentsare selected on general combining ability, sometimes by test crosses ortopcrosses, more generally by polycrosses. Parental seed lines may bedeliberately inbred (e.g. by selfing or sib crossing). However, even ifthe parents are not deliberately inbred, selection within lines duringline maintenance will ensure that some inbreeding occurs. Clonal parentswill, of course, remain unchanged and highly heterozygous.

Whether a synthetic can go straight from the parental seed productionplot to the farmer or must first undergo one or two cycles ofmultiplication depends on seed production and the scale of demand forseed. In practice, gasses and clovers are generally multiplied once ortwice and are thus considerably removed from the original synthetic.

While mass selection is sometimes used, progeny testing is generallypreferred for polycrosses, because of their operational simplicity andobvious relevance to the objective, namely exploitation of generalcombining ability in a synthetic.

The number of parental lines or clones that enters a synthetic varieswidely. In practice, numbers of parental lines range from 10 to severalhundred, with 100-200 being the average. Broad based synthetics formedfrom 100 or more clones would be expected to be more stable during seedmultiplication than narrow based synthetics.

Hybrids.

A hybrid is an individual plant resulting from a cross between parentsof differing genotypes. Commercial hybrids are now used extensively inmany crops, including corn (maize), sorghum, sugar beet, sunflower andbroccoli. Hybrids can be formed in a number of different ways, includingby crossing two parents directly (single cross hybrids), by crossing asingle cross hybrid with another parent (three-way or triple crosshybrids), or by crossing two different hybrids (four-way or double crosshybrids).

Strictly speaking, most individuals in an out breeding (i.e.,open-pollinated) population are hybrids, but the term is usuallyreserved for cases in which the parents are individuals whose genomesare sufficiently distinct for them to be recognized as different speciesor subspecies. Hybrids may be fertile or sterile depending onqualitative and/or quantitative differences in the genomes of the twoparents. Heterosis, or hybrid vigor, is usually associated withincreased heterozygosity that results in increased vigor of growth,survival, and fertility of hybrids as compared with the parental linesthat were used to form the hybrid. Maximum heterosis is usually achievedby crossing two genetically different, highly inbred lines.

The production of hybrids is a well-developed industry involving theisolated production of both the parental lines and the hybrids whichresult from crossing those lines. For a detailed discussion of thehybrid production process, see, e.g., Wright, Commercial Hybrid SeedProduction 8:161-176, In Hybridization of Crop Plants.

Bulk Segregation Analysis (BSA).

BSA, a.k.a. bulked segregation analysis, or bulk segregant analysis, isa method described by Michelmore et al (Michelmore et al., 1991,Identification of markers linked to disease-resistance genes by bulkedsegregant analysis: a rapid method to detect markers in specific genomicregions by using segregating populations. Proceedings of the NationalAcademy of Sciences, USA, 99:9828-9832) and Quarrie et al. (Quarrie etal., Bulk segregant analysis with molecular markers and its use forimproving drought resistance in maize. 1999, Journal of ExperimentalBotany, 50(337):1299-1306).

For BSA of a trait of interest, parental lines with certain differentphenotypes are chosen and crossed to generate F2, doubled haploid orrecombinant inbred populations with QTL analysis. The population is thenphenotyped to identify individual plants or lines having high or lowexpression of the trait. Two DNA bulks are prepared, one from theindividuals having one phenotype (e.g., resistant to pathogen), and theother from the individuals having reversed phenotype (e.g., susceptibleto pathogen), and analyzed for allele frequency with molecular markers.Only a few individuals are required in each bulk (e.g., 10 plants each)if the markers are dominant (e.g., RAPDs). More individuals are neededwhen markers are co-dominant (e.g., RFLPs). Markers linked to thephenotype can be identified and used for breeding or QTL mapping.

Gene Pyramiding.

The method to combine into a single genotype a series of target genesidentified in different parents is usually referred as gene pyramiding.A non-limiting example of gene pyramiding scheme is shown in FIG. 3. Thefirst part of a gene pyramiding breeding is called a pedigree and isaimed at cumulating one copy of all target genes in a single genotype(called root genotype). The second part is called the fixation steps andis aimed at fixing the target genes into a homozygous state, that is, toderive the ideal genotype (ideotype) from the root genotype. Genepyramiding can be combined with marker assisted selection (MAS, seeHospital et al., 1992, 1997a, and 1997b, and Moreau et al, 1998) ormarker based recurrent selection (MBRS, see Hospital et al., 2000).

RNA Interference (RNAi)

RNA interference (RNAi) is the process of sequence-specific,post-transcriptional gene silencing or transcriptional gene silencing inanimals and plants, initiated by double-stranded RNA (dsRNA) that ishomologous in sequence to the silenced gene. The preferred RNA effectormolecules useful in this invention must be sufficiently distinct insequence from any host polynucleotide sequences for which function isintended to be undisturbed after any of the methods of this inventionare performed. Computer algorithms may be used to define the essentiallack of homology between the RNA molecule polynucleotide sequence andhost, essential, normal sequences.

The term “dsRNA” or “dsRNA molecule” or “double-strand RNA effectormolecule” refers to an at least partially double-strand ribonucleic acidmolecule containing a region of at least about 19 or more nucleotidesthat are in a double-strand conformation. The double-stranded RNAeffector molecule may be a duplex double-stranded RNA formed from twoseparate RNA strands or it may be a single RNA strand with regions ofself-complementarity capable of assuming an at least partiallydouble-stranded hairpin conformation (i.e., a hairpin dsRNA or stem-loopdsRNA). In various embodiments, the dsRNA consists entirely ofribonucleotides or consists of a mixture of ribonucleotides anddeoxynucleotides, such as RNA/DNA hybrids. The dsRNA may be a singlemolecule with regions of self-complementarity such that nucleotides inone segment of the molecule base pair with nucleotides in anothersegment of the molecule. In one aspect, the regions ofself-complementarity are linked by a region of at least about 3-4nucleotides, or about 5, 6, 7, 9 to 15 nucleotides or more, which lackscomplementarity to another part of the molecule and thus remainssingle-stranded (i.e., the “loop region”). Such a molecule will assume apartially double-stranded stem-loop structure, optionally, with shortsingle stranded 5′ and/or 3′ ends. In one aspect the regions ofself-complementarity of the hairpin dsRNA or the double-stranded regionof a duplex dsRNA will comprise an Effector Sequence and an EffectorComplement (e.g., linked by a single-stranded loop region in a hairpindsRNA). The Effector Sequence or Effector Strand is that strand of thedouble-stranded region or duplex which is incorporated in or associateswith RISC. In one aspect the double-stranded RNA effector molecule willcomprise an at least 19 contiguous nucleotide effector sequence,preferably 19 to 29, 19 to 27, or 19 to 21 nucleotides, which is areverse complement to the RNA of nucleic acid sequences coding forEG261, homologs of EG261, orthologs of EG261 and/or paralogs of EG261,and/or fragments and variations thereof or an opposite strandreplication intermediate. In one embodiment, said double-stranded RNAeffector molecules are provided by providing to a soybean or otherplant, plant tissue, or plant cell an expression construct comprisingone or more double-stranded RNA effector molecules. In one embodimentthe expression construct comprises a double-strand RNA derived from anyone of nucleic acid sequences coding for EG261, homologs of EG261,orthologs of EG261 and/or paralogs of EG261, and/or fragments andvariations thereof. In other embodiments, the expression constructcomprises a double-strand RNA derived from more than one sequences ofnucleic acid sequences coding for EG261, homologs of EG261, orthologs ofEG261 and/or paralogs of EG261, and/or fragments and variations thereof.In further embodiments, the expression construct comprises adouble-strand RNA derived from more than one sequences of nucleic acidsequences coding for EG261, homologs of EG261, orthologs of EG261 and/orparalogs of EG261, and/or fragments and variations thereof, and one ormore other genes involved in pathogen resistance. One skilled in the artwill be able to design suitable double-strand RNA effector moleculebased on the nucleotide sequences of nucleic acid sequences coding forEG261, homologs of EG261, orthologs of EG261 and/or paralogs of EG261,and/or fragments and variations thereof in the present invention.

In some embodiments, the dsRNA effector molecule of the invention is a“hairpin dsRNA”, a “dsRNA hairpin,” “short-hairpin RNA” or “shRNA”,i.e., an RNA molecule of less than approximately 400 to 500 nucleotides(nt), or less than 100 to 200 nt, in which at least one stretch of atleast 15 to 100 nucleotides (e.g., 17 to 50 nt, 19 to 29 nt) is basedpaired with a complementary sequence located on the same RNA molecule(single RNA strand), and where said sequence and complementary sequenceare separated by an unpaired region of at least about 4 to 7 nucleotides(or about 9 to about 15 nt, about 15 to about 100 nt, about 100 to about1000 nt) which forms a single-stranded loop above the stem structurecreated by the two regions of base complementarity. The shRNA moleculescomprise at least one stem-loop structure comprising a double-strandedstem region of about 17 to about 500 bp; about 17 to about 50 bp; about40 to about 100 bp; about 18 to about 40 bp; or from about 19 to about29 bp; homologous and complementary to a target sequence to beinhibited; and an unpaired loop region of at least about 4 to 7nucleotides, or about 9 to about 15 nucleotides, about 15 to about 100nt, about 250-500 bp, about 100 to about 1000 nt, which forms asingle-stranded loop above the stem structure created by the two regionsof base complementarity. It will be recognized, however, that it is notstrictly necessary to include a “loop region” or “loop sequence” becausean RNA molecule comprising a sequence followed immediately by itsreverse complement will tend to assume a stem-loop conformation evenwhen not separated by an irrelevant “stuffer” sequence.

The expression construct of the present invention comprising DNAsequence which can be transcribed into one or more double-stranded RNAeffector molecules can be transformed into a plant, wherein thetransformed plant produces different fatty acid compositions than theuntransformed plant. The target sequence to be inhibited by the dsRNAeffector molecule include, but are not limited to, coding region, 5′ UTRregion, 3′ UTR region of fatty acids synthesis genes. In one embodiment,the target sequence is from one or more nucleic acid sequences codingfor EG261, homologs of EG261, orthologs of EG261 and/or paralogs ofEG261, and/or fragments and variations thereof.

The effects of RNAi can bee both systemic and heritable in plants. Inplants, RNAi is thought to propagate by the transfer of siRNAs betweencells through plasmodesmata. The heritability comes from methylation ofpromoters targeted by RNAi; the new methylation pattern is copied ineach new generation of the cell. A broad general distinction betweenplants and animals lies in the targeting of endogenously producedmiRNAs; in plants, miRNAs are usually perfectly or nearly perfectlycomplementary to their target genes and induce direct mRNA cleavage byRISC, while animals' miRNAs tend to be more divergent in sequence andinduce translational repression. Detailed methods for RNAi in plants aredescribed in David Allis et al (Epigenetics, CSHL Press, 2007, ISBN0879697245, 9780879697242), Sohail et al (Gene silencing, by RNAinterference: technology and application. CRC Press. 2005, ISBN0849321417, 9780849321412), Engelke et al. (RAN Interference, AcademicPress, 2005, ISBN 0121827976, 9780121827977), and Dorm et al. (RNAInterference: Methods for Plants and Animals. CABI, 2009, ISBN1845934105, 9781845934101), which are all herein incorporated byreference in their entireties for all purposes.

The present invention provides methods of producing soybeans or otherplants containing altered and/or increased levels of pathogen toleranceand/or resistance. Such methods comprise utilizing the soybean or otherplants comprising the chimeric genes as described above.

The present invention also provides methods of breeding soybean andother plants producing altered and/or increased levels of pathogentolerance and/or resistance. In one embodiment, such methods comprise:

i) making a cross between the soybean or other plant species withnucleic acid sequences coding for EG261, homologs of EG261, ortho ofEG261 and/or paralogs of EG261, and/or fragments and variations thereofas described above to a second soybean or other plant species to make F1plants;

ii) backcrossing said F1 plants to said second soybean or plant species,respectively;

iii) repeating backcrossing step until said nucleic acid sequences areintegrated into the genome of said second soybean or other plantspecies, respectively. Optionally, such method can be facilitated bymolecular markers.

The present invention provides methods of breeding species close toGlycine max, wherein said species produces altered and/or increasedlevels of pathogen tolerance and/or resistance. In one embodiment, suchmethods comprise

i) making a cross between the non-Glycine max species containing nucleicacid sequences coding for homologs of EG261, orthologs of EG261 and/orparalogs of EG261, and, or fragments and variations thereof as describedabove to Glycine max to make F1 plants;

ii) backcrossing said F1 plants to Glycine max;

iii) repeating backcrossing step until said nucleic acid sequences areintegrated into the genome of Glycine max. Special techniques (e.g.,somatic hybridization) may be necessary in order to successfullytransfer a gene from non-Glycine max species to Glycine max. Optionally,such method can be facilitated by molecular markers.

The present invention is further illustrated by the following examplesthat should not be construed as limiting. The contents of allreferences, patents, and published patent applications cited throughoutthis application, as well as the Figures, are incorporated herein byreference in their entirety for all purposes.

EXAMPLES Example 1 Identification and Characterization of EG261 inDomesticated Soybean (Glycine max)

This invention involves the discovery that the soybean gene referred toherein as ‘EG261’ codes for a dirigent protein that confers resistanceto pathogens, including resistance to nematodes. There are 2 copies ofEG261 in soybeans, but only one of the gene copies, which is referred toherein as “copy A,” is positively selected. The second copy, which isreferred to herein as “copy B,” is well-conserved and there is noevidence that it plays any role in nematode resistance (or in any othertrait of agronomic interest). Unless otherwise specified herein, allreferences to EG261 are referring to copy A of the gene.

EG261 is very highly expressed in roots, as might be expected for a genethat confers resistance to SCN, as this is a primary route for SCNentrance to the plant.

It appears that EG261 maps into a predicted location (i.e., a QTL) onsoybean chromosome for SCN resistance. This QTL is supported by twodifferent studies (Vergel C. Concibido, Brian W. Diers, and Prakash R.Arelli Crop Science, VOL. 44, JULY-AUGUST 2004, 1121-1131, and Pin Yue,David A. Sleper and Prakash R. Arelli, Crop Science, VOL. 41,SEPTEMBER-OCTOBER 2001, 1589-1595). However, this QTL is broad, andlikely contains hundreds of genes, so the exact gene-of-interest has notpreviously been isolated. That positively-selected EG261 appears to fallwithin this QTL is consistent with our hypothesis that EG controls andconfers SCN resistance. (It is noted, however, that the QTL location isstill only tentatively mapped due to differences in size and structureof mapping populations, DNA marker platforms, and limited availabilityof DNA markers.)

EG261 soybean sequences follow. Initiation codons shown in underlinedlower case font. Termination codons shown in bolded lower case font. TheUTR sequence is in lower case.

The cDNA sequence of EG261 (SEQ ID NO: 1), derived from cultivated soybean (Glycine max)  follows:agcaagagatgctcaaacccaaattcacctacttcgatgcttcccccaccttataaattcttgcattaattaccactttcatcacccaatccacttcctctaattgacacccacctaaaccatgGCTTCCCACTTCCTCAAATCCCTCCTTCTCCTCTCCACCTATGCCCTCACCATCTCAGCAGAATACACAGGCTTCGTGGGAACACTAGACCCAAAGTCCATAGGCATACACCACAAGAAAACCTAAGCCACTTCAGGTTCTACTGGCACGAAGTCTTCAGCGGAGAAAACCCCACATCGGTTAGAATCATTCCCTCACTCCCCAAATACAACGCAACCACAACCTTCGGCTCCGTTGGAATCTTTGACACCCCTTTAACCGTGGGACCTGAGGTGTACTCCAAGGTTGTCGGAAAAGCCGAGGGCTTGTTTGCCTCCACGTCACAAACGCAGTTTGACCTGTTACTGATTTACAACTTCGCGTTGACCCAAGGGAAGTACAACGGCAGCACCATCACGTTCACGGGGAGGAGCCCCCTCTCGGAGAAGGTGAGGGAGCTGCCCATTGTTGGTGGTAGTGGGGTCTTCAAATTTGCCACTGGGTATGTTGAGTCTAGGACGCTAAGTTTTGATCCCCAAACAAGGAATAACACGGTTCAGTTCGACGTGTATATTTACTATtgatgattattgaatgtgtttttttcatgttgatgcgttatcgctttggtctgtctcacctagtttct  aEG261 coding sequence (cultivated soybean;  Glycine max. SEQ ID NO: 2):atgGCTTCCCACTTCCTCAAATCCCTCCTTCTCCTCTCCACCTATGCCCTCACCATCTCAGCAGAATACACAGGCTTCGTGGGAACACTAGACCCAAAGTCCATAGGCATACACCACAAGAAAACCCTAAGCCACTTCAGGTTCTACTGGCACGAAGTCTTCAGCGGAGAAAACCCCACATCGGTTAGAATCATTCCCTCACTCCCCAAATACAACGCAACCACAACCTTCGGCTCCGTTGGAATCTTTGACACCCCTTTAACCGTGGGACCTGAGGTGTACTCCAAGGTTGTCGGAAAAGCCGAGGGCTTGTTTGCCTCCACGTCACAAACGCAGTTTGACCTGTTACTGATTTACAACTTCGCGTTGACCCAAGGGAAGTACAACGGCAGCACCATCACGTTCACGGGGAGGAGCCCCCTCTCGGAGAAGGTGAGGGAGCTGCCCATTGTTGGTGGTAGTGGGGTCTTCAAATTTGCCACTGGGTATGTTGAGTCTAGGACGCTAAGTTTTGATCCCCAAACAAGGAATAACACGGTTCAGTTCGACGTGTATATTTACTAT tgaThe EG261 cDNA sequence is more than 99% identical to Sequences 65260 and 33011 of U.S. Pat. No. 7,569,389, each of which are identical to each other (SEQ ID NO: 3):   1atcacccaat ccacttcctc taattgacac ccacctaaac  catggcttcc cacttcctca  61aatccctcct tctcctctcc acctatgccc tcaccatctc  agcagaatac acaggcttcg 121tgggaacact agacccaaag tccataggca tacaccacaa  gaaaacccta agccacttca 181ggttctactg gcacgaagtc ttcagcggag aaaaccccac  atcggttaga atcattccct 241cactccccaa atacaacgca accacaacct tcggctccgt  tggaatcttt gacacccctt 301taaccgtggg acctgaggtg tactccaagg ttgtcggaaa  agccgagggc ttgtttgcct 361ccacgtcaca aacgcagttt gacctgttac tgatttacaa  cttcgcgttg acccaaggga 421agtacaacgg cagcaccatc acgttcacgg ggaggagccc  cctctcggag aaggtgaggg 481agctgcccat tgttggtggt agtggggtct tcaaatttgc  cactgggtat gttgagtcta 541ggacgctaag ttttgatccc caaacaagga ataacacggt  tcagttcgac gtgtatattt 601actattgatg attattgaaa gtgttttttt catgttgatg  cgttatcgct ttggtctgtc 661tcacctagtt tctacataag tttcctcttt tgagggcatg  tggtgtcatg gaataaagtc 721atctttgggc aaaaaaaaaa aaaaaaaa

Example 2 Identification of Orthologs of EG261 in Wild Soybean Species

Using analysis techniques derived from molecular evolutionary biology,gene sequences from the cultivated (i.e., domesticated) soybean, Glycinemax, were compared to orthologous genes from species of wild soybeanthat are relatives to G. max and that were suspected to be resistant toattack by the Soybean Cyst Nematode (SCN). This analysis was performedin high-throughput fashion, applying Evolutionary Genomics' proprietarysoftware and patented approach. See, e.g., U.S. Pat. No. 6,228,586; U.S.Pat. No. 6,274,319; and U.S. Pat. No. 6,280,953.

Most varieties of cultivated soybeans are susceptible to one or moreraces (now more commonly known as HG groups) of SCN, and even thosesoybean cultivars bred for resistance to SCN are now often losing thatresistance as the cyst nematodes evolve the ability to evade the currentresistance mechanisms. Present resistance mechanisms all depend upononly one or two genes, so are relatively easy targets for the evolutionof resistance.

Wild soybean relatives were chosen for analysis; these species werethose that the scientific literature suggested were tolerant to one ormore races of SCN. These putatively SCN-resistant species were thentested in standard assays for SCN susceptibility/resistance. Multipleraces (HG groups) were used to test each wild soybean accession. Inmultiple replicates, multiple plants of each wild soybean species werechallenged by SCN; each challenge involved multiple SCN races/HG groups.

After demonstrating that several wild species were indeed resistant toSCN, these species were grown to maturity in order to collect RNA. TotalRNA was prepared from individual plants of each of 5 SCN-resistantspecies. RNA was pooled from several tissues from each individual plantin order to maximize how comprehensively the transcriptome of thatspecies was sampled. RNA was used to create cDNA libraries which wereused to sequence the species' transcriptome. This was accomplished byRoche 454 high-throughput next-gen sequencing, although an anyappropriate DNA sequencing method could have been used.

The resulting ESTs were compared on a gene-by-gene basis. The homologoussequences were analyzed to identify those that have nucleic acidsequence differences between the two species. Then molecular evolutionanalysis was conducted to evaluate quantitatively and qualitatively theevolutionary significance of the differences between orthologous genesderived from cultivated soybean (G. max) and each wild species (forexample, Glycine tabacina) on a pairwise basis. Each wild species wasalso compared in pairwise fashion to every other wild soybean species.Automated bioinformatics analysis was then applied to each pairwisecomparison and only those sequences that contain a nucleotide change (orchanges) that yield evolutionarily significant change(s) were retainedfor further analysis. This enabled the identification of genes that haveevolved to confer some evolutionary advantage as well as theidentification of the specific evolved changes.

Any of several different molecular evolution analyses or Ka/Ks-typemethods can be employed to evaluate quantitatively and qualitatively theevolutionary significance of the identified nucleotide changes betweenhomologous gene sequences from related species. Kreitman and Akashi(1995) Annu. Rev. Ecol. Syst. 26:403 422; Li, Molecular Evolution,Sinauer Associates, Sunderland, Mass. 1997. For example, positiveselection on proteins (i.e., molecular-level adaptive evolution) can bedetected in protein-coding genes by pairwise comparisons of the ratiosof nonsynonymous nucleotide substitutions per nonsynonymous site (Ka) tosynonymous substitutions per synonymous site (Ks) (Li et al., 1985; Li,1993). Any comparison of Ka and Ks may be used, although it isparticularly convenient and most effective to compare these twovariables as a ratio. Sequences are identified by exhibiting astatistically significant difference between Ka and Ks using standardstatistical methods.

Preferably, the Ka-Ks analysis by Li et al. is used to carry out thepresent invention, although other analysis programs that can detectpositively selected genes between species can also be used. Li et al.(1985) Mol. Biol. Evol. 2:150 174; Li (1993); see also J. Mol. Evol.36:96 99; Messier and Stewart (1997) Nature 385:151 154; Nei (1987)Molecular Evolutionary Genetics (New York, Columbia University Press).The Ka/Ks method, which comprises a comparison of the rate ofnon-synonymous substitutions per non-synonymous site with the rate ofsynonymous substitutions per synonymous site between homologousprotein-coding regions of genes in terms of a ratio, is used to identifysequence substitutions that may be driven by adaptive selections asopposed to neutral selections during evolution. A synonymous (“silent”)substitution is one that, owing to the degeneracy of the genetic code,makes no change to the amino acid sequence encoded; a non-synonymoussubstitution results in an amino acid replacement. The extent of eachtype of change can be estimated as Ka and Ks, respectively, the numbersof synonymous substitutions per synonymous site and non-synonymoussubstitutions per non-synonymous site. Calculations of Ka/Ks may beperformed manually or by using software. An example of a suitableprogram is MEGA (Molecular Genetics Institute, Pennsylvania StateUniversity).

For the purpose of estimating Ka and Ks, either complete or partialprotein-coding sequences are used to calculate total numbers ofsynonymous and non-synonymous substitutions, as well as non-synonymousand synonymous sites. The length of the polynucleotide sequence analyzedcan be any appropriate length. Preferably, the entire coding sequence iscompared, in order to determine any and all significant changes.Publicly available computer programs, such as Li93 (Li (1993) J. Mol.Evol. 36:96 99) or INA, can be used to calculate the Ka and Ks valuesfor all pairwise comparisons. This analysis can be further adapted toexamine sequences in a “sliding window” fashion such that small numbersof important changes are not masked by the whole sequence. “Slidingwindow” refers to examination of consecutive, overlapping subsections ofthe gene (the subsections can be of any length).

The comparison of non-synonymous and synonymous substitution rates iscommonly represented by the Ka/Ks ratio. Ka/Ks has been shown to be areflection of the degree to which adaptive evolution has been at work inthe sequence under study. Full length or partial segments of a codingsequence can be used for the Ka/Ks analysis. The higher the Ka/Ks ratio,the more likely that a sequence has undergone adaptive evolution and thenon-synonymous substitutions are evolutionarily significant. See, forexample, Messier and Stewart (1997).

Ka/Ks ratios significantly greater than unity strongly suggest thatpositive selection has fixed greater numbers of amino acid replacementsthan can be expected as a result of chance alone, and is in contrast tothe commonly observed pattern in which the ratio is less than or equalto one. Nei (1987); Hughes and Nei (1988) Nature 335:167 170; Messierand Stewart (1994) Current Biol. 4:911 913; Kreitman and Akashi (1995)Ann. Rev. Ecol. Syst. 26:403 422; Messier and Stewart (1997). Ratiosless than one generally signify the role of negative, or purifyingselection: there is strong pressure on the primary structure offunctional, effective proteins to remain unchanged.

All methods for calculating Ka/Ks ratios are based on a pairwisecomparison of the number of nonsynonymous substitutions pernonsynonymous site to the number of synonymous substitutions persynonymous site for the protein-coding regions of homologous genes fromrelated species. Each method implements different corrections forestimating “multiple hits” (i.e., more than one nucleotide substitutionat the same site). Each method also uses different models for how DNAsequences change over evolutionary time. Thus, preferably, a combinationof results from different algorithms is used to increase the level ofsensitivity for detection of positively-selected genes and confidence inthe result.

It is understood that the methods described herein could lead to theidentification of soybean polynucleotide sequences that are functionallyrelated to soybean protein-coding sequences. Such sequences may include,but are not limited to, non-coding sequences or coding sequences that donot encode proteins. These related sequences can be, for example,physically adjacent to the soybean protein-coding sequences in thesoybean genome, such as introns or 5′- and 3′-flanking sequences(including control elements such as promoters and enhancers). Theserelated sequences may be obtained via searching a public genome databasesuch as GenBank or, alternatively, by screening and sequencing anappropriate genomic library with a protein-coding sequence as probe.Methods and techniques for obtaining non-coding sequences using relatedcoding sequence are well known to one skilled in the art.

After candidate genes were identified, the nucleotide sequences of thegenes in each orthologous gene pair were carefully verified by standardDNA sequencing techniques and then Ka/Ks analysis was repeated for eachcarefully sequenced candidate gene pair.

More specifically to this Example, the software ran through all possiblepairwise comparisons between putative orthologs of every gene fromcultivated soybean, G. max, as compared to the likely orthologs from thewild species, looking for high Ka/Ks ratios. The software BLASTed (inautomated fashion) every mRNA sequence from cultivated soybean againstevery sequence in the transcriptome that was sequenced from a wildrelative, for example, G. pescadrensis. The software then performedKa/Ks analysis for each gene pair (i.e., each set of ‘orthologs’),flagging the gene pairs with high Ka/Ks scores. The software thencompared every G. max sequence against every sequence of another wildrelative, for example, G. tabacina, again by doing, a series of BLASTs,and then sifting through for high Ka/Ks scores. It thus does this forthe transcriptome sequence of all the wild species in succession. Thisgives a set of candidates (see below) for subsequent analysis.

The software next compared every gene sequence in the transcriptome ofG. pescadrensis against every sequence of G. tabacina, again doingBLASTs, etc. It thus ultimately compared all of the expressed genesrepresented in the utilized cDNA libraries of every soybean speciesagainst all the genes of every other soybean species, both wild andcultivated, the goal being to find every gene that shows evidence ofpositive selection. While software was used to accomplish this analysisit can be accomplished by one skilled in the art using a calculator.i.e., it can all be “done by hand”—the software simply speeds things up.

The flagged gene pairs that emerged were then individually and carefullyre-sequenced in the lab to check the accuracy of the originalhigh-throughput reads; thus false positives were eliminated.

Next, every remaining candidate gene pair with a high Ka/Ks score wasexamined to determine if the comparison was truly orthologous, or justan artifactual false positive caused by a paralogous comparison.

Following are provided the nucleotide sequences of the orthologous genesthat were definitely, positively selected based on this analysis.Information about the geographic locations from which each accession wasoriginally collected, as well any other available data about eachaccession are also given below.

Glycine microphylla (SEQ ID NO: 4):ttaattcatcactttcatcacccaatccact:cctctaattaacacccaccaacaccatgGCTTCCCACTTCCTCAAATCCCTCCTTCTCCTCTCCACCTATGCCCTCACCATCTCAGCAGAATACACAGGCTTCGTGGGGACACTAGACCCAAAGTCCATAGGCATACACCACAAGAAAACCCTAAGCCACTTCAGGTTCTACTGGCACGAAGTCTTCAGCGGAGAAAACCCCACAACGGTTAGAATCATTCCCTCACTCCCCAAATACAACACAACCACAACCTTCGGTTCCGTTGGAATCTTTGACAACACTTTAACCGTGGGACCTGAGGTGTACTCCAAGGTTGCCGGAAAAGCCGAGGGCTTGTTTGCCTCCACGTCACAAACGCAGTTTAACCTGTTACTGATTTACAGCTTCGCGTTGACCCAAGGGAAGTACAACGGCAGCACCATCACGTTCACGGGGAGGAGCCCCCTCTCGGAGAAGGTGAGGGAGCTGCCCATTGTTGGTGGCAGTGGGGTCTTCAAATTTGCCACTGGGTATGTTGAGTCTAGGACGCTAAGTTTTGATCCCCAAACGAGGAATAACACGGTTCAGTTCGACGTGTATATTTACTATtgatgattattgaatgtgttttttacatgttgatgtgttagagctttggtgtgtgtcacttagtttcta

This sequence is from Accession P1505196 which was collected 30 Jul.1983. Queensland, Australia. Locality: 5.4 km from road junction(Evelyn) towards Tumoulin. Latitude: 17 deg 32 min S (−17.53333333),Longitude: 145 deg 26 min E (145.433333333) Elevation: 900 meters.

Demonstrated to be resistant to SCN race 3 (Bauer, S., T. Hymowitz, andG. R. Noel. 2007. Soybean cyst nematode resistance derived from Glycinetomentella in amphiploid (G. max×G. tomentella) hybrid lines.Nematropica 37:277-285).

Glycine pescadrensis (SEQ ID NO: 5):ataaattcttgcattaattcatcactttcatcacccaatccatttcctctaattaacacccaccaaaaccatgGCTTCCCACTTCCTCAAATCCCTCCTTCTCCTCTCCACCTATGCCCTCACCATCTCAGCAGAATACACAGGCTTCGTGGGCACACTAGACCCAAAGTCCATAGGCATACACCACAAGAAAACCCTAAACCACTTCAGGTTCTACTGGCACGAAGTCTTCAGCGGAGAAAACCCCACATCGGTTAGAATCATTCCCTCACTCCCCAAATACAACACAACCACAACCTTCGGTTCCGTTGGAATCTCTGACAACGCTTTAACCGTGGGACCTGAGGTGTACTCCAAGGTTGTCGGAAAAGCCGAGGGGTTGTTTGTCTCCACGTCACAAACGCAGTTTGACCTGTTACTGATTTACAACTTCGCGTTGACCCAAGGGAAGTACAACGGCAGCACCATCACGTTCACGGGGAGGAGGCCCCTCTCGGAGAAGGTGAGGGAGCTGCCCATTGTAGGTGGCAGTGGGGTCTTCAAATTTTCCACTGGGTATGTTGAGTCTAGGACGCTAAGTTTTGATCCCCAAACGAGGAATAACACGGTTCAGTTCGACGTGTATATTTACTATtgatgattattgaatgtgttttttccatgttgatgtgttatagctttggtatgtgtcacttagtttcta

This sequence is from Accession PI537287, which was collected 7 Apr.1988. Taiwan. Locality: Military area, Sea Stride (Penghu Bridge),Paisha Island, Pescadores Islands. Latitude: 23 deg 40 min 0 sec N(23.66666667). Longitude: 119 deg 34 min 48 sec E (119.58). Elevation: 5meters.

Glycine tabacina (New South Wales, Australia,  SEQ ID NO: 6):catcacccaatccact:cctctaattaacacccaccaacaccatgGCTTCCCACTTCCTCAAATCCCTCCTTCTCCTCTCCACCTATGCCCTCACCATCTCAGCAGAATACACAGGCTTCGTGGGGACACTAGACCCAAAGTCCATAGGCATACACCACAAGAAAACCCTAAGCCACTTCAGGTTCTACTGGCACGAAGTCTTCAGCGGAGAAAACCCCACAACGGTTAGAATCATTCCCTCACTCCCCAAATACAACACAACCACAACCTTCGGTTCCGTTGGAATCTTTGACAACACTTTAACCGTGGGACCTGAGGTGTACTCTAAGGTTGCCGGAAAAGCCGAGGGCTTGTTTGCCTCCACGTCACAAACGCAGTTTAACCTGTTACTGATTTACAGCTTCGCGTTGACCCAAGGGAAGTACAACGGCAGCACCATCACGTTCACGGGGAGGAGCCCCCTCTCGGAGAAGGTGAGGGAGCTGCCCATTGTTGGTGGCAGTGGGGTCTTCAAATTTGCCACTGGGTATGTTGAGTCTAGGACGCTAAGTTTTGATCCCCAAACGAGGAATAACACGGTTCAGTTCGACGTGTATATTTACTATtgatgattattgaatgtgttttttacatgttgatgtgttagagctttggtgt gtgtcacttagtttcta

This sequence is from G. tabacina Accession PI446968, collected in: NewSouth Wales, Australia, and some time prior to 1980. Locality: Towardwest Wyalong 35 km from Condobolin. Latitude: 33 deg 20 min S(−33.33333333), Longitude: 147 deg 8 min E (147.13333333) Elevation: 260meters.

Demonstrated to be resistant to SCN race 3 (Bauer, S., T. Hymowitz, andG. R. Noel. 2007. Soybean cyst nematode resistance derived from Glycinetomentella in amphiploid (G. max×G. tomentella) hybrid lines.Nematropica 37:277-285).

Glycine tabacina (Taiwan, SEQ ID NO: 7):cctacttcgatgcttcccccaccttataaattcttgcattaattcatcactttcatcacccaatccatttcctctaattaacacccaccaaaaccatgGCTTCCCACTTCCTCAAATCCCTCCTTCTCCTCTCCACCTATGCCCTCACCATCTCAGCAGAATACACAGGCTTCGTGGGCACACTAGACCCAAAGTCCATAGGCATACACCACAAGAAAACCCTAAACCACTTCAGGTTCTACTGGCACGAAGTCTTCAGCGGAGAAAACCCCACATCGGTTAGAATCATTCCCTCACTCCCCAAATACAACACAACCACAACCTTCGGTTCCGTTGGAATCTCTGACAACGCTTTAACCGTGGGACCTGAGGTGTACTCCAAGGTTGTCGGAAAAGCCGAGGGCTTGTTTGTCTCCACGTCACAAACGCAGTTTGACCTGTTACTGATTTACAACTTCGCGTTGACCCAAGGGAAGTACAACGGCAGCACCATCACGTTCACGGGGAGGAGCCCCCTCTCGGAGAAGGTGAGGGAGCTGCCCATTGTAGGTGGCAGTGGGGTCTTCAAATTTTCCACTGGGTATGTTGAGTCTAGGACGCTAAGTTTTGATCCCCAAACGAGGAATAACACGGTTCAGTTCGACGTGTATATTTACTATtgatgattattgaatgtgttttttccatgttgatgtgttatagctt tggtatgtgtcacttagtttcta

This sequence is from G. tabacina Accession PI559281, collected inPenghu, Taiwan. 8 Apr. 1988. Locality: Airport (NE) on Chimei Island.Latitude: 23 deg 33 min 48 sec N (23.5633577) Longitude: 119 deg 37 min41 sec E (119.628067).

Demonstrated to be resistant to SCN race 3 (Bauer, S., T. Hymowitz, andG. R. Noel. 2007. Soybean cyst nematode resistance derived from Glycinetomentella in amphiploid (G. max×G. tomentella) hybrid lines.Nematropica 37:277-285.)

Glycine tomentella (An alternate name for this species, used in some literature, is G. latifolia) (Australia, SEQ ID NO: 8):cacctacttcgatgcttccccccaccttataaattcttgcattaattcatcactttcatcacccaatccatttcctctaattaacacccaccaaaaccatgGCTTCCCACTTCCTCAAATCCCTCCTTCTCCTCTCCACCTATGCCCTCACCATCTCAGCAGAATACACAGGCTTCGTGGGCACACTAGACCCAAAGTCCATAGGCATACACCACAAGAAAACCCTAAACCACTTCAGGTTCTACTGGCACGAAGTCTTCAGCGGAGAAAACCCCACATCGGTTAGAATCATTCCCTCACTCCCCAAATACAACACAACCACAACCTTCGGTTCCGTTGGAATCTCTGACAACGCTTTAACCGTGGGACCTGAGGTGTACTCCAAGGTTGTCGGAAAAGCCGAGGGCTTGTTTGTCTCCACGTCACAAACGCAGTTTGACCTGTTACTGATTTACAACTTCGCGTTGACCCAAGGGAAGTACAACGGCAGCACCATCACGTTCACGGGGAGGAGCCCCCTCTCGGAGAAGGTGAGGGAGCTGCCCATTGTAGGTGGCAGTGGGGTCTTCAAATTTTCCACTGGGTATGTTGAGTCTAGGATGCTAAGTTTTGATCCCCAAACGAGGAATAACACGGTTCAGTTCGACGTGTATATTTACTATtgatgattattgaatgtgttttttccatgttgatgtgttatagctttggtatgtgtcacttagtttctatataagtttccama 

This sequence is from G. tomentella Accession PI 499946, collected inNew South Wales, Australia. Locality: Delungra. Latitude: 29 deg 39 min0 sec S (−29.65), Longitude: 150 deg 50 min E (150.83333333) Elevation:600 meters.

Demonstrated to be resistant to SCN (Bauer, S., T. Hymowitz, and G. R.Noel. 2007. Soybean cyst nematode resistance derived from Glycinetomentella in amphiploid (G. max×G. tomentella) hybrid lines.Nematropica 37:277-285).

All of the above EG261 sequences from the wild relatives of G. max areall “copy A” (as given above for G. max). Copy A from each wild specieshas been strongly positively selected as compared to copy A of G. max).In addition, when compared to each other (i.e., not compared only tocultivated soybeans) EG261 copy A from each wild species examined hasbeen strongly positively selected relative to each of the other wildspecies. This suggests that each wild species has likely been confrontedby several different races of SCN, as well as by different species andeven categories of pathogens (such as bacteria, fungi, insects, andnematodes) so that each wild species of soybean has thus likelyevolved/achieved resistance to multiple SCN races.

Our demonstration of the clear episodes of positive selection in thewild soybean species, relative to each of the other wild species,suggests strongly that the orthologs of EG261 from each of these wildspecies is of value for creating SCN resistance in cultivated soybeans.

Example 3 Characterization of Orthologs of EG261 in Wild Soybean Species

Plants from wild, related species containing the orthologs identifiedaccording to Example 2 were assayed for their actual ability to conferresistance. In this example, wild soybean species of the genus Glycinewere tested for their innate ability to resist challenge by soybean cystnematode (SCN). Germplasm accessions were obtained from the NationalPlant Germplasm System (NPGS).

Seeds were geminated and seedlings were tested for SCN resistance inconical containers. (Niblack, T., Tylka, G. L., Arelli, P., Bond, J.,Diers, B., Donald. P., Faghihi, J., Ferris, V. R., Gallo, K., Heinz, R.D., Lopez-Nicora, H., Von Qualen, R., Welacky, T., and Wilcox, J. 2009.A standard greenhouse method for assessing soybean cyst nematoderesistance in soybean: SCE08 (standardized cyst evaluation 2008).Online. Plant Health Progress doi:10.1094/PHP-2009-0513-01-RV.)

For those species that were confirmed to have innate ability to resistSCN, RNAs from several tissues (including root, stem, and leaf) wereflash-frozen and both genomic DNA and RNA were prepared from the frozentissues. RNAs from multiple tissues from each plant then were pooled forsubsequent construction of cDNA libraries (one library per species).High-throughput 454 Titanium sequencing was performed for each cDNAlibrary. It is noted, however, that any suitable high-throughputsequencing format would have been acceptable and using this particularmethod was not critical to the success of these experiments.

The resulting transcriptome sequence datasets for each species wereanalyzed in order to identify any positively selected transcripts usingproprietary analysis software of Evolutionary Genomics, Inc.

Candidate genes were verified using protocols in transgenic soybeanplants that test for effectiveness in protecting the transformed soybeanplants from attack by the SCN organism as well as the Phytophthorafungus. We report here results from experiments where RNAi is used toknock out EG261 in an SCN-resistant line (Fayette). After knockout, thenormally resistant Fayette line became SCN sensitive. The control forthis experiment was RNAi silencing of EG261 in the Williams 82, which isSCN-sensitive: no difference in sensitivity was observed after silencingof EG261 in this line. These experiments are interpreted to mean thatresistance to SCN is conferred by the EG261 gene; if one knocks out theEG261 allele in SCN-resistant lines, the result is that SCN resistanceis then lost. (Interestingly, the allele of EG261 in the SCN-sensitiveWilliam 82 line is already either non-functional or at least not veryeffective in conferring SCN-resistance, so that silencing of the EG261allele does not change the SCN-sensitive phonotype of Williams 82.)Importantly, the resistant line (Fayette) is one that was created bycross-breeding to soybean land-races that display SC N-resistance. (Itis believed that the land-races became SCN-resistant because ofintrogression with wild SCN-resistant soybean species.)

Example 4 Silencing of EG261 Eliminates the SCN-Resistant Phenotype inSCN-Resistant Soybeans

Construct pG2RNAi2 was produced carrying a hairpin to silence the targetgene EG261 and the constructs were used to produce transgenic roots inthe soybean variety ‘Fayette.’ Two replications of SCN demographicsexperiments were conducted for the ‘Fayette’ transgenic roots. For adetailed description of the test protocol used herein, see Melito et al.(2010) A nematode demographics assay in transgenic roots reveals nosignificant impacts of the Rhg1 locus LRR-Kinase on soybean cystnematode resistance, BMC Plant Biology 10:104, 14 pages.

Approximately 250 hatched J2 nematodes were placed near the tip of eachtransgenic soybean root, with root identities blinded by assignment ofrandomized numbers. After two weeks, roots were fixed and stained withacid fuchsin and the number and growth stage of all nematodes within orattached to each root was assessed (average 76 total nematodes perroot).

In the first of two replicate experiments, in the ‘Fayette’ transgenicroots, expression levels of EG261 are on average about 12% of Fayettewild-type. 11 of 14 transgenic roots had silencing >50% and wereincluded: 3 roots were excluded due to poor silencing. In the secondreplicate experiment, tissue was saved but silencing was not checked.Therefore, all of the data was included (hence the phenotypic impact ofsilencing may be masked a bit due to inclusion of some non-silencedroots in the data that was obtained).

Data show significant shift toward susceptibility (FIG. 1). Bars showthe proportion of nematode population on each root that advanced pastthe J2 stage (mean std.error). F: Fayette (SCN-resistant); W: Williams82 (SCN-susceptible): EV: transformed with empty vector; hp: transformedwith EG261 hairpin gene silencing construct; Ox: transformed withconstruct expressing Glynma01g31770 under control of strong G. maxubiquitin promoter. ANOVA P value for similarity of means is 0.005 for FEV compared to F hp, and 0.97 for W EV compared to W Ox.

The control for this experiment was RNAi silencing of EG261 in thesoybean cultivar Williams 82, which is SCN-sensitive: no difference insensitivity was observed after silencing of EG261 in this line. Theseexperiments were interpreted to mean that resistance to SCN is conferredby the EG261 gene; if one knocks out the EG261 allele in SCN-resistantlines, the result is that SCN resistance is then lost. However, theallele of EG261 in the sensitive William 82 line is already eithernon-functional or at least not very effective in conferringSCN-resistance, so that silencing of the EG261 allele does not changethe SCN-sensitive phonotype of Williams 82. Importantly, the resistantline (Fayette) is one that was created by cross-breeding to soybeanland-races that display SCN-resistance. (It is believed that theland-races became SCN-resistant because of introgression with wildSCN-resistant soybean species.) This is strong evidence for the role ofEG261 in influencing/controlling SCN resistance in soybeans.

Example 5 Transformation of Tomato with EG261

‘Big Boy’ tomato plants can be transformed with a construct containingEG261 using standard transformation technology for tomato plants. See,e.g., Antonio Di Matteo, Maria Manuela Rigano, Adriana Sacco, LuigiFrusciante and Amalia Barone (2011). Genetic Transformation in Tomato:Novel Tools to Improve Fruit Quality and Pharmaceutical Production,Genetic Transformation, Prof. Maria Alvarez (Ed.), ISBN:978-953-307-364-4, InTech.

Transformation of tomato plants with a construct containing EG261 can beaccomplished with either the cultivated/the wild soybean orthologsincluded in this patent, or any of the other EG261 orthologs describedin this application, including, the tomato ortholog (SEQ ID NO 16).

The transformed tomato plants can be tested for disease resistance ascompared to the untransformed control ‘Big Boy’ tomato plants.

Diseases which can be tested for conferred or enhanced pathogentolerance and/or resistance as a result of such transformation includeroot rot caused by Rhizoctonia solani, Fusarium solani andSclerotiumrolfsii. For procedures used to test for tolerance/resistanceto these root diseases see, e.g., Abd-El-Kareem, F., Nehal S. El-Mougy,Nadia G. El-Gamal and Y. O. Fotouh (2006) Use of Chitin and ChitosanAgainst Tomato Root Rot Disease under Greenhouse Conditions Research,Journal of Agriculture and Biological Sciences, 2(4): 147-152.

The transformed tomato plants showing conferred and/or enhancedresistance to one or more of these root diseases can be tested toconfirm the presence of EG261 by using one or more of the followingprocedures: (a) isolating nucleic acid molecules from said plant andamplifying sequences homologous to the EG261 polynucleotide; (b)isolating nucleic acid molecules from said plant and performing aSouthern hybridization to detect the EG261 polynucleotide; (c) isolatingproteins from said plant and performing a Western Blot using antibodiesto a protein encoded by the EG261 polynucleotide: and/or (d)demonstrating the presence of mRNA sequences derived from a EG261polynucleotide mRNA transcript and unique to the EG261 polynucleotide.

Example 6 Tomato Breeding with Tomato Plants Expressing EG261

A ‘Big Boy’ tomato plant with a conferred copy of the coding sequencefor EG261 as obtained in Example 6 can be crossed to a plant of the‘Early Girl’ variety and the resultant progeny can be tested fortolerance/resistance to the three root diseases.

The presence of the EG261 polynucleotide can be confirmed in theresultant ‘Early Girl’ progeny according to the procedures set forth inExample 5.

In a further procedure, the transformed ‘Early Girl’ tomato plant can bebackcrossed one or more times to ‘Early Girl’ to produce a near isogenicor isogenic ‘Early Girl’ tomato with the coding sequence for EG261.

Example 7 Transformation of Maize with EG261

‘B73’ and/or ‘Mo17’ inbred maize plants can be transformed with aconstruct containing EG261 using standard transformation technology formaize. See, e.g., Sidorov and Duncan, 2008 (Agrobacterium-Mediated MaizeTransformation: Immature Embryos Versus Callus, Methods in MolecularBiology, 526:47-58); Frame et al., 2002 (Agrobacteriumtumefaciens-Mediated Transformation of Maize Embryos Using a StandardBinary Vector System, Plant Physiology, May 2002, Vol. 129, pp. 13-22):and, Ahmadabadi et al. 2007 (A leaf-based regeneration andtransformation system for maize (Zea mays L.), Transgenic Res. 16,437-448).

Transformation of maize plants with a construct containing EG261 can beaccomplished with either the cultivated/the wild soybean orthologsincluded in this patent, or with the maize ortholog of these EG261genes.

The transformed maize plants can be tested for nematode/pest resistanceas compared to the untransformed, control ‘B73’ or ‘Mo17’ maize plants.For procedures used to test for tolerance/resistance to nematodes see,e.g., Sasser, et al. (1984) Standardization of Host Suitability Studiesand Reporting of Resistance to Root-Knot Nematodes, Crop NematodeResearch & Control Project (CNRCP), a cooperative publication of TheDepartment of Plant Pathology, North Carolina State University and theUnited States Agency for International Development, North Carolina StateUniversity Graphics, Raleigh, N.C., 10 pages; and, Cook, R. and K. Evans(1987) Resistance and Tolerance, In: Principles and practice of nematodecontrol in plants, R. H. Brown and B. R. Kerry (editors), pg. 179-231.

The transformed maize plants showing conferred and/or enhancedresistance to one or more races of nematodes can be tested to confirmthe presence of EG261 by using one or more of the following procedures:(a) isolating nucleic acid molecules from said plant and amplifyingsequences homologous to the EG261 polynucleotide; (b) isolating nucleicacid molecules from said plant and performing a Southern hybridizationto detect the EG261 polynucleotide: (c) isolating proteins from saidplant and performing a Western Blot using antibodies to a proteinencoded by the EG261 polynucleotide; and/or (d) demonstrating thepresence of mRNA sequences derived from a EG261 polynucleotide mRNAtranscript and unique to the EG261 polynucleotide.

The transformed maize plants with confirmed tolerance and/or resistanceto one or more nematode races can be maintained by self-pollinating theplants, harvesting the resultant seed and growing the resistant plantsfrom such harvested seed.

Example 8 Maize Breeding with Maize Plants Expressing EG261

A ‘B73’ or ‘Mo17’ corn inbred plant with a conferred copy of the codingsequence for EG261 as obtained in Example 7 can be crossed to atransformed or non-transformed inbred ‘Mo17’ ‘B73’ plant, respectively,and the resultant hybrid F1 progeny can be tested fortolerance/resistance to nematodes.

The presence of the EG261 polynucleotide can be confirmed in theresultant hybrid progeny according to the procedures set forth inExample 7.

In a further procedure, the transformed hybrid maize plant can bebackcrossed one or more times to its recurrent parent (i.e., either‘B73’ or ‘Mo17’ as applicable) to produce a near isogenic or isogenicmaize inbred with the coding sequence for EG261.

Example 9 EG261 Overexpression

Overexpression of EG261 was tested using two replications of the soybeanvariety ‘Williams 82.’ For a detailed description of the test protocolused herein see Melito et al. (2010) A nematode demographics assay intransgenic roots reveals no significant impacts of the Rhg1 locusLRR-Kinase on soybean cyst nematode resistance, BMC Plant Biology10:104, 14 pages.

Expression level in “overexpression roots” was found to be 4.3-foldgreater, however native EG261 gene is expressed very highly, only 5-foldless than ubiquitin, so these expression levels appear to be consistentwith expectations.

Data show no significance difference from normal ‘Williams 82’ (i.e.,without overexpression of EG261), but these inconclusive results areprobably because expression levels were not as rigorously controlled aswas desirable.

Example 10 Identification of Orthologs of EG261 in Other Plant Species

Using analysis techniques derived from molecular evolutionary biology,gene sequences from the cultivated (i.e., domesticated) soybean, Glycinemax, were used to identify orthologous genes from other plant species.The following table provides the SEQ ID Nos. of the nucleotide sequencesof genes that are orthologous to the positively selected soybean EG261copy A gene. Predicted amino acid sequences, and information about thegeographic locations from which each accession was originally collected,as well any other available information about each accession are alsogiven below.

Nucleotide Amino acid Geographic locations sequence sequence andaccession Plant species SEQ ID NO. SEQ ID NO. information Solanumlycopersicum (or 16 25 cultivated tomato Solanum esculentum) Solanumperuvianum 17 26 Peruvian tomato, a.k.a., Peruvian nightshade Solanumchmielewskii 18 27 Solanum habrochaites 19 28 Solanum corneliomulleri 2029 Capsicum annuum 21 30 chili pepper Capsicum annuum 22 31 hot pepperSolanum melongena 23 32 Eggplant Solanum tuberosum 24 33 PotatoPhaseolus vulgaris 34 37 Common bean varieties “Blue Lake” (a bush typesnap bean that was developed from the Blue Lake Pole bean), HendersonBush Lima Bean (bean variety dates to 1885), Dixie Speckled ButterpeaLima Bean, Hidatsa Red Indian, and Kabouli Black Garbanzo Asian Bean(collected in Kabul, Afghanistan) Phaseolus vulgaris 35 38 Common beanvariety Scarlet Emperor (dates at least to 1750, was originally fromCentral America) Phaseolus vulgaris 36 39 Jacob's Cattle Bean Coffeaarabica 40 43 coffee plant, Allele A Coffea arabica 41 44 coffee plant,Allele B Capsicum annuum 42 45 cultivated pepper (“ornamental” pepper)Zea mays 46 51 Sorghum sp. 47 52 Oryza sativa, 48 53 Triticum sp. 49 54Hordeum sp. 50 55 Gossypium hirsutum 56 57 Variety FJ600364 Gossypiumhirsutum 58 59 Variety FJ600365 64 65 Vigna unguiculata (Cowpea) 60 61Monkey tail (collected in Africa) Vigna unguiculata (Cowpea) 62 63 Pea,varieties Risina Del Trasiorfino (collected in Perugia, Italy) andShanty Pea (collected in South Carolina, USA)

Example 11 Characterization of Orthologs of EG261 Isolated fromAdditional Plant Species

The gene orthologs from wild, related species can be assayed for theirability to confer resistance. (In this example, corresponding wild plantspecies can also be tested for their innate ability to resist challengeby one or more parasitic nematodes. Seeds are germinated and seedlingsare tested for resistance to one or more parasitic nematodes by anysuitable standard testing methods.)

For each species examined, the species-specific ortholog of EG261 can beanalyzed for evidence of positive selection, using proprietary analysissoftware of Evolutionary Genomics, Inc. Orthologs that appear to havebeen positively selected can then be assayed for the ability to conferresistance as described for soybean (G. max) in Examples 3 and 4.

Example 12 Silencing of EG261 Eliminates the Nematode ResistantPhenotype in Nematode-Resistant Plants

Constructs are produced carrying an RNAi silencing DNA fragment (e.g., ahairpin, a double strand, an antisense, an inverted repeat, etc.)targeting each one of the EG261 orthologs isolated in Example 10. Theseconstructs are used to produce transgenic plants for each plant species.

Hatched J2 nematodes are placed near the tip of each transgenic plant.After a time sufficient for nematode infection, roots are fixed andstained with acid fuchsin and the number and growth stage of allnematodes within or attached to each root is assessed.

Data shows significant shift toward susceptibility in one or moretransgenic plant species compared to their corresponding wild-typenematode-resistant control plant species.

Example 13 Introducing EG261 Orthologs into Heterologous Plant Species

In some embodiments, the EG261 orthologs conferring nematode resistanceisolated according to the present invention from one plant species canbe introduced into a nematode-susceptible plant of the same species inorder to create nematode resistance into the plant.

However, in some other embodiments, the EG261 orthologs of the presentinvention isolated from one plant species can be introduced into anematode-susceptible plant of different species of the same genus, or aplant species of different genus in order to introduce nematoderesistance into the plant.

For example, a gene encoding any one of the proteins of SEQ ID NOs: 9-15which were isolated from soybean can be also introduced into other plantspecies, such as Solanaceae species (e.g., Solanum lycopersicum, Solanumchemielewskii, Solanum habrochaites, Solanum corneliomulleri, Capsicumannuum, Solanum melongena, Solanum tuberosum), Phaseolus vulgaris, Vignaunguiculata, Coffee arabica, Zea mays, Sorghum spp., Oryza sativa,Triticum spp. Hordeum spp., Gossypium hirsutum, and Heliotropiumcurassavicum. Similarly, a gene encoding anyone of the proteins of SEQID NOs: 25, 26, 27, 28, 29, 30, 31, 32, 33, 38, 39, 43, 44, 45, 51, 52,53, 54, 55, 57, 59, 61, 63, 64, and 65 can be introduced into otherplant species.

Following the methods described in Example 11, plant species comprisinga heterologous EG261 gene or an ortholog thereof are created and tested.The results will confirm that one or more EG261 genes or orthologsthereof can introduce nematode resistance when heterologously expressedin a plant species different from the plant species from which theheterologous EG261 genes or orthologs thereof were isolated.

Example 14 Introducing Nematode Resistance into Nematode-SusceptiblePlants

This example shows that a gain-of-SCN-resistance phenotype in apreviously SCN-sensitive soybean cultivar was observed when the EG261ortholog from a wild SCN-resistant soybean species (Glycinepescadrensis) was transformed into the SCN-sensitive ‘Williams 82’soybean cultivar. That is, by using transformation (knock-in) acultivated SCN-sensitive soybean cultivar becomes SCN resistant. Infact, the level of resistance achieved is roughly equivalent to that ofthe only known commercially-available SCN-resistant soybean cultivar.The results indicate that, by using an EG261 ortholog isolated from anematode resistant plant to transform a plant that one can achieve SCNsuppression, to near zero in some cases, in that plant.

More specifically, EG261 orthologs from 2 species, (G. pescadrensis andG. microphylla) were tested.

The G. pescadrensis EG261 ORF with 5′ and 3′ UTR was PCR amplified fromcDNA and ligated into pCR8. A NOS terminator was cloned intopSM101-Gmubi to create an over-expression construct with a strongpromoter and terminator. Alternative promoters for root expression asdiscussed elsewhere herein or are well known to those skilled in the artcould be used instead. The G. pescadrensis EG261 ORF was excised frompCR8 (5′ PstI, 3′ BamHI) and ligated into pSM101-Gmubi-NOS. VectorpSM101-Gmubi-G. pescadrensis EG261-NOS was verified by sequencing.

Similarly, the G. microphylla EG261 ORE with UTR was PCR-amplified fromcDNA and ligated into pCR8. A NOS terminator was cloned intopSM101-Gmubi to create an over-expression construct with a strongpromoter and terminator. The G. microphylla EG261 ORF was excised frompCRS (5′ PstI, 3′ BamHI) and ligated into pSM101-Gmubi-NOS. VectorpSM101-Gmubi-G. microphylla EG261-NOS was verified by sequencing.

These constructs were transformed into Agrobacterium rhizogenes.Cotyledons of SCN-susceptible genotype (‘Williams 82’) and SCN-resistantgenotype (‘Fayette’) were germinated and then transformed withAgrobacterium containing these EG261 over-expression constructs. Hairyroots emerged from these cotyledons and GFP positive roots (thoseexpressing the genes of interest as well as an empty control vector)were harvested and subcultured (approximately 10 roots per genotype witheach construct). SCN cysts were crushed and eggs hatched to produceinfective juveniles (J2). Approximately 220 sterile J2 were placed ontothese transgenic hairy roots.

The second-stage juvenile, or J2, is the only life stage that soybeancyst nematode can penetrate roots. The first-stage juvenile occurs inthe egg, and third- and fourth-stages occur in the roots. The J2 entersthe root moving through the plant cells to the vascular tissue where itfeeds. The J2 induces cell division in the root to form specializedfeeding sites. As the nematode feeds, it swells. The female swells somuch that her posterior end bursts out of the root and she becomesvisible to the naked eye. In contrast, the adult male regains a wormlikeshape, and he leaves the root in order to find and fertilize the largefemales. The female continues to feed as she lays 200 to 400 eggs in ayellow gelatinous matrix, forming an egg sac which remains inside her.She then dies and her cuticle hardens forming a cyst. The eggs may hatchwhen conditions in the soil are favorable, the larvae developing insidethe cyst and the biological cycle repeating itself. There are usuallythree generations in the year. In the autumn or in unfavorableconditions, the cysts containing dormant larvae may remain intact in thesoil for several years. The juvenile molts to the J3 and beginsenlarging as the reproductive system develops. Nematodes which becomefemales are no longer able to leave the root. They continue to enlargeas they go through the J3 and J4 stages. During this time, cells aroundthe head of the nematode enlarge to form nurse cells or giant cells. Forroot-knot nematode, galls will typically develop on the root. Uponbecoming adults, root-knot nematodes will begin to lay eggs (up toseveral hundred) which are contained in a gelatinous matrix at theposterior end of the body. The egg mass may be within the root or partlyor wholly exposed on the root surface while the swollen body of thefemale remains within the root. Therefore, we count the 52 and J3numbers to assess the extent of the nematode infestation.

As mentioned above, variety ‘Williams’ was used as a SCN susceptiblecultivar and variety ‘Fayette’ was used as a SCN-resistant control.Experimental results from three independent experiments are provided inthe table below. The testing methods used herein are described in Melitoet al. (BMC Plant Biology, 2010, 10:104, incorporated herein byreference in its entirety. In the following table, “EV” refers to ‘emptyvector’, i.e., a control plant that has been transformed with a vectorconstruct that lacks the experimental gene but that otherwise consistsof the complete vector. “OX” refers to ‘over-expression’, i.e., thoseexperimental plants in which the vector contains the experimental EG261ortholog, and the experimental knock-in gene is actively expressed athigh levels. “W” refers to the SCN-sensitive Williams 82 soybeancultivar, while “F” refers to the normally SCN-resistant soybeancultivar. “Microph” refers to the EG261 ortholog derived from G.microphylla, while “Pesc” refers to the EG261 ortholog derived from G.pescadrensis.

Sample Sample Description J2 J3 (and >+ Males) Notes (J3 & Above/Total)Independent Experiment #1 1 W-pescOX 32 13 0.289 2 W-pescOX 38 7 0.156 3W-microphOX 8 9 0.529 4 W-EV 24 19 3 males 0.442 5 W-pescOX 11 3 0.214 6W-EV 14 19 0.576 7 W-microphOX 19 12 Confusing 0.387 8 F-EV 20 3 2 males0.130 9 W-EV 16 25 0.610 10 W-EV 22 27 2 males 0.551 11 W-EV 8 6 0.42912 W-microphOX 10 9 0.474 13 W-pescOX 30 10 6 males 0.250 14 W-EV 38 40.095 15 W-pescOX 22 5 0.185 16 W-pescOX 50 41 0.451 17 W-microphOX 3721 0.362 18 W-pescOX 32 13 0.289 19 W-microphOX 4 9 0.692 20 W-microphOX18 20 0.526 21 W-EV 4 16 0.800 22 W-microphOX 5 20 0.800 23 W-microphOX24 F-EV 6 5 Faint stain 0.455 25 F-EV 32 16 0.333 26 W-microphOX 8 160.667 27 F-EV 8 9 Faint stain 0.529 28 W-EV 9 5 0.357 29 F-EV 8 11 0.57930 W-microphOX 14 5 0.263 31 W-microphOX 10 23 8 males 0.697 32 F-EV 409 0.184 33 W-pescOX 3 10 0.769 34 W-EV 8 13 0.619 35 W-EV 11 5 Faint -Not 0.313 confidant at all 36 W-EV 3 7 0.700 37 W-EV 2 9 0.818 38 F-EV 57 0.583 39 W-microphOX 3 10 0.769 40 W-microphOX 7 7 0.500 41 W-EV 4 110.733 42 W-microphOX 8 6 Faint, hard to 0.429 count 43 F-EV 27 9 0.25044 W-EV 6 11 0.647 45 W-EV 15 9 0.375 46 F-EV 67 20 0.230 IndependentExperiment #2 1 W-pescOX 25 11 0.306 2 F-EV 48 27 0.360 3 W-pescOX 44 630.589 4 W-pescOX 57 53 0.482 5 W-pescOX 35 51 Very 0.593 susceptible 6W-microphOX 31 25 0.446 7 F-EV 54 53 0.495 8 W-EV 37 58 Looks kind of0.611 susceptible? Recount? 9 F-EV 49 15 0.234 10 W-microphOX 64 800.556 11 F-EV 60 16 0.211 12 W-pescOX 20 37 0.649 13 F-EV 53 72 0.576 15W-microphOX Contaminated 16 W-EV 27 55 0.671 17 W-pescOX 28 37 0.569 18W-microphOX 30 48 0.615 19 W-EV 54 31 0.365 20 W-pescOX 46 49 0.516 21W-EV 21 42 Hard to count 0.667 22 F-EV 94 23 0.197 23 W-pescOX 66 250.275 24 W-pescOX 46 47 0.505 25 W-microphOX 14 14 0.500 26 W-EV 14 400.741 27 W-microphOX 95 37 0.280 28 W-pescOX 27 24 0.471 29 W-EV 15 390.722 30 F-EV 30 69 0.697 31 F-EV 86 26 Hard to count 0.232 32 W-EV 5357 0.518 33 W-EV 58 30 0.341 34 F-EV 96 41 0.299 35 W-EV 82 24 0.226 36W-microphOX 86 53 Hard to count 0.381 37 W-pescOX 68 49 0.419 38 W-EV 5365 0.551 39 W-microphOX 89 86 0.491 40 F-EV 76 34 0.309 41 W-EV 44 380.463 42 W-EV 82 53 0.393 43 F-EV Contaminated 44 F-EV 59 43 0.422 45W-pesoOX 80 39 0.328 46 W-microphOX 45 85 0.654 47 W-pescOX 85 44 0.341Independent Experiment #3 1 W-EV 24 11 0.314285714 2 F-EV Contaminated 3F-EV 39 15 0.277777778 4 W-pescOX 24 13 0.351351351 5 W-pescOX 26 40.133333333 6 F-EV 34 15 0.306122449 7 W-pescOX 32 20 0.384615385 8W-microphOX 17 14 0.451612903 9 W-EV 7 11 0.611111111 10 W-pescOX 19 110.366666667 11 W-EV 21 12 0.363636364 12 W-pescOX 31 20 0.392156863 13W-pescOX 11 9 0.45 14 W-EV 17 11 0.392857143 15 W-EV 10 7 0.411764706 16W-EV 17 11 0.392857143 17 F-EV 37 8 0.177777778 18 W-EV 27 13 0.325 19F-EV 38 13 0.254901961 20 F-EV 40 23 0.365079365 21 W-microphOX 15 210.583333333 22 W-microphOX 18 11 0.379310345 23 W-microphOX 21 300.588235294 24 W-EV 4 9 0.692307692 25 W-pescOX 16 14 0.466666667 26W-microphOX 12 12 0.5 27 W-pescOX 19 23 0.547619048 28 W-microphOX 32 300.483870968 29 F-EV 40 20 0.333333333 30 W-EV 19 22 0.536585366 31 F-EV8 12 0.6 32 W-pescOX 11 14 0.56 33 W-EV 17 15 0.46875 34 F-EV 28 150.348837209 35 W-EV Less than 5 Nematodes 36 W-microphOX 15 190.558823529 37 F-EV 10 7 0.411764706 38 W-pescOX 14 6 0.3 39 F-EV 19 110.366666667 40 W-microphOX 10 17 0.62962963 41 W-microphOX 15 17 0.5312542 W-EV 14 11 0.44 43 W-microphOX 19 23 0.547619048 44 W-microphOX 18 300.625 45 W-microphOX 22 11 0.333333333 46 W-pescOX 12 8 0.4 47 F-EV 2418 0.428571429 J3 & Above/Total normalized to W-EV within eachexperiment SE F-EV 0.722940836 0.05183849 W-EV 0.999999999 0.051787021W-microOX 1.04598414 0.046093455 W-pescOX 0.824925891 0.052329931

Examination of the results make clear that transformation with the EG261ortholog from G. pescadrensis causes the normally SCN-sensitive Williams82 cultivar to acquire a level of SCN-resistance that approximates thatof the SCN-resistant cultivar Fayette. Although the G. microphyllaortholog didn't appear to show obvious SCN suppression in the initialassessment, this may not rule out the effectiveness of the G.microphylla EG261 ortholog given the number of variables that can affecttransformation experiments. (We are repeating this first-pass experimentfor the G. microphylla ortholog.) In contrast, the EG261 ortholog fromG. pescadrensis is clearly capable of conferring resistance to SCN whenintroduced into SCN-sensitive soybean plants. We are currentlyinvestigating the effectiveness of EG261 orthologs from additional wildsoybean relatives that are SCN tolerant.

Example 15 Transformation of Beans and Cowpeas with EG261

Other agriculturally relevant species such as beans (Phaseolus vulgaris)and cowpeas (Vigna unguiculata) can also be transformed with constructscontaining EG261 using standard transformation technology for beans andcowpeas respectively. See, e.g., Brasileiro et al., 1996 (Suceptibilityof common and tepary beans to Agrobacterium spp. Strains and improvementof Agrobacterium-mediated transformation using microprojectilebombardment, J. Am. Soc. Hortic. Sci. 121, 810-815); Estrada-Navarreteet al., 2007 (Fast, efficient and reproducible genetic transformation ofPhaseolus spp. by Agrobacterium rhizogenes, Nat. Protoc 2007;2(7):1819-24); Ivo et al., 2008 (Biolistic-mediated genetictransformation of cowpea (Vigna unguiculata) and stable Mendelianinheritance of transgenes, Plant Cell Rep 27:1475-83); Popelka et al.,2006 (Genetic transformation of cowpea (Vigna unguiculata L.) and stabletransmission of the transgenes to progeny, Plant Cell Rep 2006;25:304-12).

Transformation of beans or cowpea plants with a construct containingEG261 can be accomplished with either the cultivated/the wild soybeanorthologs included in this patent, or with their respective bean(Phaseolus vulgaris), or cowpea (Vigna unguiculata) orthologs of theseEG261 genes.

The transformed bean plants can be tested for nematode/pest resistanceas compared to the untransformed, control ‘Condor,’ ‘Matterhorn,’‘Sedona,’ ‘Olathe,’ or ‘Montcalm’ bean plants. The transformed cowpeaplants can also be tested for nematode/pest resistance as compared tothe untransformed, control ‘IT 82E-16,’ ‘TVu 1390,’ or ‘B301’ cowpeaplants. For procedures used to test for tolerance/resistance tonematodes see, e.g., Sasser, et al. (1984) Standardization of HostSuitability Studies and Reporting of Resistance to Root-Knot Nematodes,Crop Nematode Research & Control Project (CNRCP), a cooperativepublication of The Department of Plant Pathology, North Carolina StateUniversity and the United States Agency for International Development,North Carolina State University Graphics, Raleigh, N.C., 10 pages; and,Cook, R. and K. Evans (1987) Resistance and Tolerance, In: Principlesand practice of nematode control in plants, R. H. Brown and B. R. Kerry(editors), pg. 179-231.

The transformed bean or cowpea plants showing conferred and/or enhancedresistance to one or more races of nematodes can be tested to confirmthe presence of EG261 by using one or more of the following procedures:(a) isolating nucleic acid molecules from said plant and amplifyingsequences homologous to the EG261 polynucleotide; (b) isolating nucleicacid molecules from said plant and performing a Southern hybridizationto detect the EG261 polynucleotide; (c) isolating proteins from saidplant and performing a Western Blot using antibodies to a proteinencoded by the EG261 polynucleotide; and/or (d) demonstrating thepresence of mRNA sequences derived from a EG261 polynucleotide mRNAtranscript and unique to the EG261 polynucleotide.

The transformed bean or cowpea plants with confirmed tolerance and/orresistance to one or more nematode races can be maintained byself-pollinating the plants, harvesting the resultant seed and growingthe resistant plants from such harvested seed.

Example 16 Improving Nematode Resistance by Introducing EG261 Genes intoNematode Resistant Plant Varieties

In some embodiments, the EG261 orthologs conferring nematode resistanceisolated according to the present invention can be introduced into anematode-susceptible plant of the same or different species in order toincrease nematode resistance of the plant.

However, in some other embodiments, the EG261 orthologs of the presentinvention can be introduced into nematode-resistant plants of the sameor different species in order to further increase nematode resistance ofthe plant.

For example, a gene encoding any one of the proteins of SEQ ID NOs:9-15, 25, 26, 27, 28, 29, 30, 31, 32, 33, 37, 38, 39, 43, 44, 45, 51,52, 53, 54, 55, 57, 59, 61, 63, 64, or 65 can be introduced intonematode resistant plants containing any nematode resistant traits suchas rhg1, rhg4, or any other known QTLs such as those listed in Concibidoet al., 2004 (A decade of QTL mapping for cyst nematode resistance insoybean, Crop Sci. 44: 1121-1131); Shannon et al., (2004) Breeding forresistance and tolerance, In: Biology and management of soybean cystnematode, 2^(nd) ed, P. Schmitt, J. A. Wrather, and R. D. Riggs,(editors). pg 155-180; and Klink et al., (2013) Engineered Soybean CystNematode Resistance, In: Soybean-Pest Resistance, Hany A. El-Shemy(editor), pg-139-172.

Following the methods described in Example 11, nematode resistant plantscomprising a heterologous EG261 gene or an ortholog thereof are createdand tested. The results will confirm that one or more EG261 genes ororthologs thereof can improve nematode resistance when expressed in aplant species with previously existing resistance to nematodes.

Example 17 Conferring Nematode Resistance into Nematode-SusceptiblePlants Via Non-Transgenic Methods

As set-forth in the Examples above, nematode resistance is can beintroduced into a nematode-susceptible plant via the genetictransformation of an EG261 gene or ortholog into the genome of saidplant.

In other embodiments, nematode resistance can be incorporated into aplant via non-transgenic (i.e., traditional) methods such as plantbreeding. For example a cross can be made between a first plant with anaturally-occurring or transgenic EG261 gene copy conferring nematoderesistance, and a second plant to produce a F1 plant. This F1 plant canthen be subjected to multiple backcrossings to generate a near isogenicor isogenic line, wherein the nematode resistant copy of EG261 isintegrated into the genome of the second plant.

For example, G. microphylla, or G. Pescadrensis plants withnaturally-occurring gene copies of EG261 can be crossed with a secondplant, to produce a F1 plant. This F1 plant can then be subjected tomultiple backcrossings to generate a near isogenic or isogenic line,wherein the nematode resistance copy of EG261 is integrated into thegenome of the second plant. Similarly, transgenic plants containingcopies of G. microphylla, or G. Pescadrensis EG261 genes can be crossedwith a second plant, to produce a F1 plant. This F1 plant can then besubjected to multiple backcrossings to generate a near isogenic orisogenic line, wherein the nematode resistance copy of EG261 isintegrated into the genome of the second plant.

Example 18 Identification of EG261 Native Promoters

Native promoters were identified by sequencing genomic DNA upstream fromeach EG261 gene variant. The sequenced upstream DNA included 5′untranslated regions as well as 5′ portions the coding regions for eachEG261 gene. Sequences obtained from the sequencing reactions of G.pescadrensis and G. max EG261 genes are disclosed in SEQ ID NO: 67 andSEQ ID NO: 68 respectively.

The sequenced polynucleotides were then analyzed with promoter analysissoftware to identify promoter regions. Each promoter analysis wasconducted in duplicate using both the “Neural Network PromoterPrediction” (NNPP) software and the “Prediction of PLANT Promoters”(TSSP) software (Reese M G, 2001. Application of a time-delay neuralnetwork to promoter annotation in the Drosophila melanogaster genome,Comput Chem 26(1), 51-6; Solovyev V. V., Salamov A. A. (1997) TheGene-Finder computer tools for analysis of human and model organismsgenome sequences. In Proceedings of the Fifth International Conferenceon Intelligent Systems for Molecular Biology eds. Rawling C., Clark D.,Altman R, Hunter L., Lengauer T., Wodak S., Halkidiki, Greece, AAAIPress, 294-302; Solovyev V. V. (2001) Statistical approaches inEukaryotic gene prediction. In Handbook of Statistical Genetics eds.Balding D. et al., John Wiley & Sons, Ltd., p. 83-127; and Solovyev V V,Shahmuradov I A. (2003) PromH: Promoters identification usingorthologous genomic sequences. Nucleic Acids Res. 31(13):3540-3545). Thenative promoter sequences were confirmed by both analysis methodsreaching the same result.

Core promoter sequences for the G. pescadrensis and G. max EG261 genesare shown in FIG. 5.

Unless defined otherwise, all technical and scientific terms herein havethe same meaning as commonly understood by one of ordinary skill in theart to which this invention belongs. Although any methods and materials,similar or equivalent to those described herein, can be used in thepractice or testing of the present invention, the preferred methods andmaterials are described herein. All publications, patents, patentpublications, and nucleic acid and amino acid sequences cited areincorporated by reference herein in their entirety for all purposes.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.

While the invention has been described in connection with specificembodiments thereof it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth and as follows in the scope ofthe appended claims.

The invention claimed is:
 1. A transgenic soybean plant, plant part,plant cell, or plant tissue culture comprising a construct comprising anucleic acid sequence encoding an EG261 polypeptide having at least 97%sequence identity to SEQ ID NO: 12; wherein said transgenic soybeanplant, or a transgenic soybean plant produced from said transgenic plantpart, plant cell, or plant tissue culture expresses said EG261polypeptide, and has enhanced tolerance and/or resistance to soybeancyst nematodes (SCN) compared to untransformed control soybean plants,and wherein the construct further comprises an EG261 native promoteroperably linked to said nucleic acid encoding the EG261 polypeptide. 2.The transgenic plant, plant part, plant cell, or plant tissue culture ofclaim 1, wherein the EG261 native promoter comprises a core promoterselected from the group consisting of SEQ ID NOs: 69 and
 70. 3. Thetransgenic soybean plant, plant part, plant cell, or plant tissueculture of claim 1, wherein the construct further comprises a genetermination sequence.
 4. The transgenic soybean plant, plant part, plantcell, or plant tissue culture of claim 3, wherein the gene terminationsequence is a nopaline synthase (NOS) terminator.
 5. The transgenicsoybean plant, plant part, plant cell, or plant tissue culture of claim2, wherein the construct further comprises a gene termination sequence.6. The transgenic soybean plant, plant part, plant cell, or plant tissueculture of claim 5, wherein the gene termination sequence is a nopalinesynthase (NOS) terminator.
 7. A method for producing a transgenicsoybean plant having enhanced tolerance and/or resistance to soybeancyst nematode, said method comprising: (i) transforming a soybean plantcell with a construct comprising a nucleic acid sequence encoding anEG261 polypeptide having at least 97% identity to SEQ ID NO: 12; and(ii) cultivating the transgenic soybean cell under conditions conduciveto regeneration and mature plant growth; wherein the transgenic soybeanplant regenerated from said transgenic plant cell expresses said EG261polypeptide, and has enhanced tolerance and/or resistance to soybeancyst nematodes compared to untransformed control soybean plants, andwherein the construct further comprises a EG261 native promoter operablylinked to said nucleic acid encoding the EG261 polypeptide.
 8. Themethod of claim 7, wherein the EG261 native promoter comprises a corepromoter selected from the group consisting of SEQ ID NOs: 69 and
 70. 9.A method of producing hybrid soybean seed, said method comprisingcrossing the transgenic soybean plant of claim 1 with another soybeanplant, and harvesting the resultant seed.
 10. Progeny plants of thesoybean plant produced by the method of claim 9, wherein the progenysoybean plants have enhanced tolerance and/or resistance to soybean cystnematodes compared to untransformed control soybean plants as a resultof inheriting the nucleic acid.
 11. A method of breeding soybean plantsto produce plants with enhanced tolerance and/or resistance to soybeancyst nematodes, said method comprising: (i) making a cross between afirst transgenic soybean plant of claim 1 with a second plant to producean F1 plant; (ii) backcrossing the F1 plant to the second plant; and(iii) repeating the backcrossing step one or more times to generate anear isogenic or isogenic line, wherein the construct of claim 1 isintegrated into the genome of the second plant and the near isogenic orisogenic line derived from the second plant with the nucleic acidsequence encoding the EG261 polypeptide has enhanced pathogen toleranceand/or resistance to soybean cyst nematodes compared to that of thesecond plant without said nucleic acid sequence.
 12. The method of claim11, wherein the soybean plant is Glycine max.
 13. The transgenic soybeanplant, plant part, plant cell, or plant tissue culture of claim 1,wherein the nucleic acid sequence encodes an EG261 polypeptide having atleast 98% sequence identity to SEQ ID NO:
 12. 14. The transgenic soybeanplant, plant part, plant cell, or plant tissue culture of claim 1,wherein the nucleic acid sequence encodes an EG261 polypeptide having atleast 99% sequence identity to SEQ ID NO:
 12. 15. The transgenic soybeanplant, plant part, plant cell, or plant tissue culture of claim 1,wherein the nucleic acid sequence encodes an EG261 polypeptide having atleast 100% sequence identity to SEQ ID NO:
 12. 16. The method forproducing a transgenic soybean plant having enhanced tolerance and/orresistance to soybean cyst nematodes of claim 7, wherein the nucleicacid sequence encodes an EG261 polypeptide having at least 98% sequenceidentity to SEQ ID NO:
 12. 17. The method for producing a transgenicsoybean plant having enhanced tolerance and/or resistance to soybeancyst nematodes of claim 7, wherein the nucleic acid sequence encodes anEG261 polypeptide having at least 99% sequence identity to SEQ ID NO:12.
 18. The method for producing a transgenic soybean plant havingenhanced tolerance and/or resistance to soybean cyst nematodes of claim7, wherein the nucleic acid sequence encodes an EG261 polypeptide havingat least 100% sequence identity to SEQ ID NO: 12.